CN108290953B

CN108290953B - anti-PD-1 antibodies and compositions

Info

Publication number: CN108290953B
Application number: CN201680069542.1A
Authority: CN
Inventors: G.加勒; M.加德; K.克福德; I.D.霍拉克; T.布奎因; M.克拉格; M.佩德森
Original assignee: Symphogen AS
Current assignee: Laboratoires Servier SAS
Priority date: 2015-10-02
Filing date: 2016-09-30
Publication date: 2022-05-31
Anticipated expiration: 2036-09-30
Also published as: CA3000564A1; RU2750675C1; TW201726734A; JP2018529359A; AU2016333517A1; EP4105235A1; US11034765B2; EP3368572B9; RS63490B1; IL258214A; PL3368572T3; HUE059788T2; AU2016333517B2; EP3368572A1; HRP20221041T1; BR112018006257A2; MY190324A; PT3368572T; SI3368572T1; ZA201801852B

Abstract

The present invention relates to anti-PD-1 antibodies and methods of using them to treat diseases and conditions associated with PD-1 activity (e.g., cancer).

Description

anti-PD-1 antibodies and compositions

Cross reference to related applications

This application claims priority to U.S. patent application 62/236,341 filed on day 2/10/2015, the disclosure of which is incorporated herein by reference in its entirety.

Sequence listing

The present application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy (created on 28/9/2016) is named 022675_ WO052_ sl.txt and is 65,000 bytes in size.

Background

PD-1 (also known as programmed cell death protein 1 and CD279) is a 268 amino acid cell surface receptor belonging to the immunoglobulin superfamily. PD-1 is a member of the CD28 family of T cell regulators and is expressed on T cells, B cells and macrophages. Which binds to ligands PD-L1 (also known as the B7 homologue) and PD-L2 (also known as B7-DC).

PD-1 is a type I membrane protein whose structure comprises an extracellular IgV domain, a transmembrane domain, and an intracellular tail containing two phosphorylation sites. PD-1, known as an immune checkpoint protein, functions as an inducible immunoregulatory receptor, playing a role in, for example, negative regulation of T cell responses to antigen stimulation.

PD-L1 is the dominant ligand for PD-1. Binding of PD-L1 to PD-1 inhibits T cell activity, reduces cytokine production, and inhibits T cell proliferation. Cancer cells expressing PD-L1 are able to take advantage of this mechanism to inactivate the anti-tumor activity of T cells via binding of PD-L1 to PD-1 receptors.

In view of its immune response modulating properties, PD-1 has been investigated as a potential target for immunotherapy, including the treatment of cancer and autoimmune diseases. Two anti-PD-1 antibodies (pembrolizumab and nivolumab) have been approved in the united states and europe for the treatment of certain cancers.

Given the key role of PD-1 as an immunomodulator, there is a need for new and improved immunotherapies targeting PD-1 to treat cancer and certain disorders of the immune system.

Summary of The Invention

The present invention is directed to novel recombinant antibodies targeting PD-1, as well as pharmaceutical compositions comprising one or more of these antibodies, and the use of the antibodies and pharmaceutical compositions for enhancing immunity in a patient, and for treating cancer derived from a tissue such as: skin, lung, intestine, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas. It is contemplated that the antibodies of the invention, whether used alone or in combination with another cancer therapy, such as an antibody targeting another immune checkpoint protein, may provide superior clinical responses compared to currently available therapies for such cancers, including antibody therapies.

In one embodiment, the invention provides an anti-PD-1 antibody, or antigen-binding portion thereof, wherein the antibody competes for binding to human PD-1 with any one of the following, or binds to the same epitope of human PD-1 as any one of the following: antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380.

In some embodiments, an anti-PD-1 antibody comprises an H-CDR1-3 comprising the H-CDR1-3 sequence of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380, respectively.

In some embodiments, the anti-PD-1 antibody has an amino acid sequence that is identical to the heavy chain variable domain (V) of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380_H) At least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) of the same V_HA domain.

In some embodiments, the anti-PD-1 antibody has a V comprising antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380_HV of amino acid sequence_H。

In some embodiments, the anti-PD-1 antibody has a V comprising antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380_HAmino acid sequence and SEQ ID No: 67 (HC) of the heavy chain constant region amino acid sequence.

In some embodiments, an anti-PD-1 antibody comprises L-CDR1-3 comprising the L-CDR1-3 sequence of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380, respectively.

In some embodiments, the anti-PD-1 antibody has an amino acid sequence corresponding to that of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378,12796.15376, 12777.15382, 12760.15375, or 13112.15380 light chain variable domain (V)_L) At least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) of the same V_LA domain.

In some embodiments, the anti-PD-1 antibody has a V comprising antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380_LV of amino acid sequence_L。

In some embodiments, the anti-PD-1 antibody has a V comprising antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380_LAmino acid sequence and SEQ ID No: 68 (LC) of a light chain constant region amino acid sequence.

In some embodiments, the anti-PD-1 antibody comprises any of the heavy chain sequences described above and any of the light chain sequences described above.

In some embodiments, an anti-PD-1 antibody comprises the H-CDR3 and L-CDR3 amino acid sequences of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380.

In some embodiments, an anti-PD-1 antibody comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380.

In some embodiments, the anti-PD-1 antibody has an amino acid sequence that is identical to the V of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380, respectively_HAnd V_LAt least 90% (e.g., at least 92%, at least 95%, at least 98%, or at least 99%) of the same V_HAnd V_L。

In some embodiments, an anti-PD-1 antibody has a sequence comprising antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.1, respectively5380V_HAnd V_LAmino acid sequences or V of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380, respectively_HAnd V_LV consisting of an amino acid sequence_HAnd V_L。

In some embodiments, the anti-PD-1 antibody has HC and LC amino acid sequences comprising or consisting of HC and LC amino acid sequences of antibody 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, or 13112.15380, respectively.

In some embodiments, an anti-PD-1 antibody has (1) a V comprising an antibody selected from the group consisting of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380_HAmino acid sequence and SEQ ID No: 67, HC of the heavy chain constant region amino acid sequence; and (2) V comprising selected antibodies_LAmino acid sequence and SEQ ID No: 68, LC of the light chain constant region amino acid sequence.

In some embodiments, an anti-PD-1 antibody or antigen-binding portion of the invention comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

a) are respectively SEQ ID No: 18. 19, 20, 21, 22, and 23;

b) are respectively SEQ ID No: 24. 25, 26, 27, 28, and 29;

c) are respectively SEQ ID No: 30. 31, 32, 33, 34, and 35;

d) are respectively SEQ ID No: 36. 37, 38, 39, 40, and 41;

e) are respectively SEQ ID No: 42. 43, 44, 45, 46, and 47;

f) are respectively SEQ ID No: 48. 49, 50, 51, 52, and 53;

g) are respectively SEQ ID No: 54. 55, 56, 57, 58, and 59; or

h) Are respectively SEQ ID No: 60. 61, 62, 63, 64, and 65.

In some embodiments, the anti-PD-1 antibodies or antigen-binding portions of the invention comprise a heavy chain variable domain and a light chain variable domain having the amino acid sequences:

a) are respectively SEQ ID No:2 and 3;

b) are respectively SEQ ID No: 4 and 5;

c) are respectively SEQ ID No: 4 and 66;

d) are respectively SEQ ID No: 6 and 7;

e) are respectively SEQ ID No: 8 and 9;

f) are respectively SEQ ID No: 10 and 11;

g) are respectively SEQ ID No: 12 and 13;

h) are respectively SEQ ID No: 14 and 15; or

i) Are respectively SEQ ID No: 16 and 17.

In some embodiments, an anti-PD-1 antibody of the invention comprises:

a) comprises SEQ ID No:2 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 3 and 68;

b) comprises SEQ ID No: 4 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 5 and 68;

c) comprises SEQ ID No: 4 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 66 and 68;

d) comprises SEQ ID No: 6 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 7 and 68;

e) comprises SEQ ID No: 8 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 9 and 68;

f) comprises SEQ ID No: 10 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 11 and 68;

g) comprises SEQ ID No: 12 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 13 and 68;

h) comprises SEQ ID No: 14 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 15 and 68; or

i) Comprises SEQ ID No: 16 and 67 and HC comprising the amino acid sequences of SEQ ID nos: 17 and 68.

In some embodiments, the antibodies or antigen binding portions of the invention comprise H-CDR1-3 and L-CDR1-3, said H-CDR1-3 and L-CDR1-3 comprising SEQ ID nos: 18-20 and SEQ ID No: 21-23. In some embodiments, the anti-PD-1 antibody comprises a heavy chain variable region comprising SEQ ID No:2 of the amino acid sequence V_HAnd a polypeptide comprising SEQ ID No:3 of the amino acid sequence V_L. In a specific embodiment, the anti-PD-1 antibody comprises a heavy chain variable region comprising SEQ ID No:2 and 67 and a light chain comprising the amino acid sequence of SEQ ID No:3 and 68.

The invention also provides anti-PD-1 antibodies, or antigen-binding portions thereof, that bind to an epitope of PD-1 that comprises amino acid residue K131 (e.g., 12819 and 12865 antibodies, such as the antibodies listed in tables 1, 4-9, and 11-14). In some embodiments, the epitope further comprises amino acid residues P130 and a132, and may additionally comprise amino acid residues V64 and L128 (e.g., 12819 antibody). In some embodiments, the epitope further comprises amino acid residue E136 (e.g., 12865 antibody).

The invention also provides a polypeptide comprising the amino acid sequence of SEQ ID No:1, and an epitope of PD-1 of T145 or an antigen-binding portion thereof (e.g., 13112 antibodies, such as those listed in 1, 4-7, 9, and 11-14).

In a specific embodiment, the antibody or portion binds to a polypeptide comprising SEQ ID No:1, an epitope of PD-1 comprising amino acid residues V64, L128, P130, K131, and a132 (e.g., 12819 antibody), and a heavy chain variable region comprising SEQ ID No:1 (e.g., 12865 antibody), or an epitope of PD-1 comprising amino acid residues K131 and E136 of SEQ ID No:1 and PD-1 of amino acid residue V44 of T145 (e.g., 13112 antibody).

The invention also provides a polypeptide comprising the amino acid sequence of SEQ ID No:1 and 122-140 or an antigen binding portion thereof (e.g., the 12819 or 12865 antibodies). In some embodiments, the monoclonal antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1, amino acid residues 56-64, 69-90, and 122-140 (e.g., the 12819 antibody). In some embodiments, the antibody or portion is identical to SEQ ID No:1 (or a fragment thereof) to (e.g., 12819 or 12865 antibodies). In some embodiments, the antibody or portion is identical to SEQ ID No: residue 136-140 of 1 (or a fragment thereof) binds (e.g., 12819 or 12865 antibody). In some embodiments, the antibody or portion is identical to SEQ ID No:1 (or fragments thereof) and residues 136-140 (or fragments thereof) (e.g., 12819 or 12865 antibodies).

In some embodiments, an anti-PD-1 antibody or antigen-binding portion of the invention has at least one of the following properties:

a) at a K of 750pM or less_DBinds to human PD-1;

b) with a K of 7nM or less_DBinds to cynomolgus monkey PD-1;

c) with a K of 1nM or less_DBinding to mouse PD-1;

d) does not bind to rat PD-1;

e) increased IL-2 secretion in SEB whole blood assay;

f) increasing IFN- γ secretion in a one-way mixed lymphocyte reaction assay;

g) inhibits the interaction between PD-1 and PD-L1 by at least 60% at a concentration of 10 μ g/ml in a flow cytometry competition assay;

h) blocking binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 μ g/ml as determined by biofilm (Bio-Layer) interferometry; and

i) inhibiting tumor growth in vivo.

Examples of such antibodies include, but are not limited to, the 12819 antibody (with properties a-i); 12748. 12892 and 12777 antibodies (having at least properties a, b, and e-h); 12865 and 12796 antibodies (having at least properties a, b, e, f, and h) and 12760 and 13112 antibodies (having at least properties a, b, e, and f). In some embodiments, the anti-PD-1 antibodies or antigen-binding portions of the invention have all of the properties described. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, and e-h. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, e, f, and h. In some embodiments, the anti-PD-1 antibody or antigen-binding portion has at least properties a, b, e, and f.

Unless otherwise indicated, 12819, 12748, 12865, 12892, 12796, 12777, 12760, and 13112 each refer to a group of antibodies having the same six CDRs and having a total of the first five digits in their ten-digit numbering nomenclature. For example, 12748 includes antibody variants 12748.15381 and 12748.16124, which have the same six CDRs (as shown in table 2). It is expected that each group of antibodies will share the same or substantially the same biological properties.

In some embodiments, the anti-PD-1 antibodies or antigen-binding portions of the invention do not compete with pembrolizumab or nivolumab for binding to PD-1. In some embodiments, the anti-PD-1 antibodies or antigen-binding portions of the invention do not bind to the same epitope as pembrolizumab or nivolumab; for example, an antibody or portion of the invention binds to one or more residues on PD-1 that are not bound by pembrolizumab or nivolumab.

In another aspect, the invention provides a pharmaceutical composition comprising at least one anti-PD-1 antibody or antigen-binding portion thereof as described herein and a pharmaceutically acceptable excipient.

The invention further provides isolated nucleic acid molecules comprising a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding a light chain or an antigen-binding portion thereof of an anti-PD-1 antibody as described herein, or both.

The invention also provides vectors comprising such isolated nucleic acid molecules, wherein the vectors further comprise an expression control sequence.

The invention also provides a host cell comprising a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof of an anti-PD-1 antibody as described herein, a nucleotide sequence encoding a light chain or an antigen-binding portion thereof of an anti-PD-1 antibody as described herein, or both.

The present invention also provides a method for producing an antibody or antigen-binding portion thereof as described herein, comprising providing a host cell comprising a nucleotide sequence encoding a heavy chain of an anti-PD-1 antibody or antigen-binding portion thereof as described herein and a nucleotide sequence encoding a light chain of an anti-PD-1 antibody or antigen-binding portion thereof as described herein, culturing the host cell under conditions suitable for expression of the antibody or portion, and isolating the resulting antibody or portion.

The invention also provides bispecific binding molecules having the binding specificity of an anti-PD-1 antibody described herein and the binding specificity of another anti-PD-1 antibody (e.g., another anti-PD-1 antibody described herein) or an antibody that targets a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease condition, such as cancer.

The present invention also provides a method for enhancing immunity in a patient in need thereof (e.g., a human patient), comprising administering to the patient an anti-PD-1 antibody or antigen-binding portion thereof, pharmaceutical composition, or bispecific binding molecule as described herein.

The invention further provides a method for treating cancer in a patient (e.g., a human patient) comprising administering to the patient an anti-PD-1 antibody or antigen-binding portion thereof, a pharmaceutical composition, or a bispecific binding molecule as described herein. In some embodiments, the cancer is derived from a tissue selected from the group consisting of: skin, lung, intestine, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus and pancreas. The cancer can be, for example, advanced or metastatic melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, or hodgkin's lymphoma. In some embodiments, the method further comprises administering a chemotherapeutic agent, an antineoplastic agent, an anti-angiogenic agent, a tyrosine kinase inhibitor, or a PD-1 pathway inhibitor.

The invention further provides an antibody or antigen-binding portion of the invention for use in the above-mentioned treatment and the use of an antibody or antigen-binding portion of the invention for the manufacture of a medicament for the above-mentioned treatment, i.e. the treatment of a human in need thereof to enhance the immune system thereof, and the treatment of a human suffering from a cancer, such as one of the above-mentioned cancers.

Brief Description of Drawings

FIG. 1 shows the V of the anti-PD-1 antibody AAS-12819 containing constant fragments cloned in-frame with the corresponding human heavy chain IgG1-CH1-CH2-CH3 and human light chain lambda, respectively_HAnd V_LPCR products of the area (shown in black). Restriction sites used for this cloning are ApaI and AvrII. The restriction sites AscI and NheI are at V_HAnd V_LShown between the 5' ends. The plasmid origin of replication is depicted as pUC ori and the gene conferring ampicillin resistance is depicted as AmpR.

FIG. 2 shows a graph having a value at V_HAnd V_LUsing a dual CMV promoter with AscI and NheI restriction sites inserted between the 5' ends of (a). V_HAnd V_LSequences are depicted in black and other annotated genetic elements are depicted in white.

Figures 3A-3C show representative flow cytometry dot plots of: (A) an antibody clone that specifically binds to cells transfected with human PD-1, (B) a clone that non-specifically binds to CHO-S cells, and (C) a clone that does not bind to any of the cell populations used in the screening.

FIG. 4 shows the frequency of lymphocytes expressing PD-1 in six donors (D1-D6) before and after stimulation with SEB (Staphylococcus enterotoxin B).

FIGS. 5A-I show titration of candidate anti-PD-1 antibodies in SEB assays.

FIGS. 6A-H show titration of candidate anti-PD-1 antibodies in a one-way MLR assay.

FIGS. 7A-B show the binding of PD-L1 to PD-1 expressing cells in the presence of anti-PD-1 antibodies.

Figure 8 shows a summary of the identified epitope groups (epitopic bins) of the tested anti-PD-1 antibodies 12866.13188, 12807.13177, 12819.17149, 12865.17150, 12892.13195, 12777.15382, 12760.13169, 13112.15380, and nivolumab and pembrolizumab analogs. Antibodies attached as black lines indicate cross-blocking activity. Antibodies were grouped according to competition patterns with other anti-PD-1 antibodies. Nivo: a nivolumab analog; pembro: pembrolizumab analogs.

FIG. 9 (panels A-G) shows the sites of antibody epitopes on the structure of human PD-1(PDB 4ZQK and 2M 2D). A) A schematic of the human PD-1 extracellular domain (ECD) (residues 33-150). The sites of the GFCC 'and ABED β sheets and the C' -D loop are elucidated. B) Human PD-1 at the same viewing angle as in (a): a sketch of the human PD-L1 complex. C) Molecular modeling of pembrolizumab epitopes shown in density plots, darker regions represent regions mediating stronger binding. The black areas represent contact residues found by alanine scanning. D) A molecular model of the epitope of nivolumab as represented in (C). E) Molecular models of the 12819 antibody epitope as represented in (C). F) Molecular models of the 12865 antibody epitope as represented in (C). G) A molecular model of the epitope of the non-ligand-blocking 13112 antibody as represented in (C).

FIG. 10 (panels A-D) shows the efficacy of treatment with anti-PD-1 antibodies 12819, 17149 or vehicle (vehicle) on tumor growth in four isogenic tumor models. A) CT26 (colon cancer). B) C38 (colon cancer). C) ASB-XIV (lung cancer). D) Sa1N (fibrosarcoma). The gray areas indicate the treatment period. Data are presented as mean ± SEM. P < 0.001.

FIG. 11 shows the anti-PD-1 antibody 12819.17149, pembrolizumab

Or vehicle treatment on tumor growth in a semi-humanized xenograft tumor model, wherein human melanoma cell line a375 was mixed with purified human CD8+ and CD4+ T cells prior to vaccination. The gray areas indicate the treatment period. Data are presented as mean ± SEM. P < 0.001.

Detailed Description

The present invention provides novel anti-human PD-1 antibodies that can be used to enhance the immune system in human patients, such as cancer patients. Unless otherwise stated, "PD-1" as used herein refers to human PD-1. Human PD-1 polypeptide sequence is available under Uniprot accession number Q15116(PDCD1 human), identified herein as SEQ ID No:1 is shown.

As used herein, the term "antibody" (Ab) or "immunoglobulin" (Ig) refers to a tetramer of two heavy (H) chains (about 50-70KDa) and two light (L) chains (about 25KDa) interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable domain (V)_H) And a heavy chain constant region (CH). Each light chain is composed of a light chain variable domain (V)_L) And a light chain constant region (CL). V_HAnd V_LDomains can be further subdivided into regions of high mutability (called "complementarity determining regions" (CDRs)) interspersed with more conserved regions (called "framework regions" (FRs)). Each V_HAnd V_LIs composed of three CDRs (H-CDR's are indicated herein as being from the heavy chain; and L-CDR's are indicated herein as being from the light chain) and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The assignment of amino acid numbers in the heavy or light chain may be based on

Definitions (Lefranc et al, Dev Comp Immunol 27 (1): 55-77 (2003)); or Kabat, Sequences of Proteins of Immunological Interest (national institute of health, Bethesda, MD (1987 and 1991)); chothia&Lesk, j.mol.biol.196: 901-917 (1987); or Chothia et al, Nature 342: 878-883(1989).

The term "recombinant antibody" refers to an antibody that is expressed from a cell or cell line that comprises a nucleotide sequence encoding the antibody, wherein the nucleotide sequence is not naturally associated with the cell.

The term "isolated protein," "isolated polypeptide," or "isolated antibody" refers to a protein, polypeptide, or antibody that, due to its derived origin or source, (1) is not associated with a naturally associated component with which it is associated in its natural state, (2) does not contain other proteins from the same species, (3) is expressed by cells from a different species, and/or (4) does not occur in nature. Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cell from which it is naturally derived will be "isolated" from the components with which it is naturally associated. Proteins can also be isolated so as to be substantially free of naturally associated components by using protein purification techniques well known in the art.

As used herein, the term "germline" refers to the nucleotide and amino acid sequence of an antibody gene and gene segment as it passes from parent to offspring via germ cells. Germline sequences are distinguished from nucleotide sequences encoding antibodies in mature B cells that have been altered by recombination and hypermutation events during the course of B cell maturation. An antibody that "utilizes" a particular germline sequence has a nucleotide or amino acid sequence that is aligned with that germline nucleotide sequence or with an amino acid sequence that is aligned closer to the specified amino acid sequence than any other germline nucleotide or amino acid sequence.

The term "affinity" refers to a measure of attraction between an antigen and an antibody. The intrinsic affinity of an antibody for an antigen is typically the binding affinity equilibrium constant (K) for a particular antibody-antigen interaction_D) And (4) showing. When K is_DAn antibody is said to bind specifically to an antigen when it is ≦ 1mM (preferably ≦ 100 nM). K_DThe binding affinity constant can be determined, for example, by surface plasmon resonance (BIAcore)^TM) Or biofilm interference (Bio-Layer interference) (e.g., using ProteOn from Bio-Rad^TMXPR36SPR System or Octet^TMSystem) measurement.

The term "k_off"refers to the off-rate constant for a particular antibody-antigen interaction. k is a radical of_offThe dissociation rate constant can be interfered with by a biological membrane (e.g., using Octet)^TMSystem) measurement.

As used herein, the term "epitope" refers to a portion (determinant) of an antigen that specifically binds to an antibody or related molecule, such as a bispecific binding molecule. Epitopic determinants generally consist of chemically active surface clusters of molecules such as amino acids or carbohydrates or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be "linear" or "conformational". In a linear epitope, all points of interaction between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In conformational epitopes, the point of interaction occurs throughout amino acid residues that are separated from each other in the primary amino acid sequence on the protein. Once the desired epitope on an antigen is determined, it is possible to generate antibodies against the epitope using techniques well known in the art. For example, antibodies directed against a linear epitope can be generated by immunizing an animal, e.g., with a peptide having the amino acid residues of the linear epitope. Antibodies directed against conformational epitopes can be generated, for example, by immunizing an animal with minidomains containing the relevant amino acid residues of the conformational epitope. Antibodies directed against a particular epitope can also be generated, for example, by immunizing an animal with the target molecule of interest or a relevant portion thereof (e.g., the ECD of PD-1), and then screening for binding to the epitope.

Whether an antibody binds to the same epitope as an anti-PD-1 antibody of the invention or competes for binding with an anti-PD-1 antibody of the invention can be determined by using methods known in the art, including but not limited to competition assays, epitope binning, and alanine scanning. In some embodiments, the test antibody and the anti-PD-1 antibody of the invention bind to at least one common residue (e.g., at least two, three, four, or five common residues) on PD-1. In a further embodiment, the contact residues on PD-1 between the test antibody and the anti-PD-1 antibody of the invention are identical. In one embodiment, an anti-PD-1 antibody of the invention is allowed to bind to PD-1 under saturating conditions and the ability of the test antibody to bind to PD-1 is then measured. If the test antibody is capable of binding to PD-1 simultaneously with the reference anti-PD-1 antibody, the test antibody binds to a different epitope than the reference anti-PD-1 antibody. However, if the test antibody is unable to bind to PD-1 simultaneously, the test antibody binds to the same epitope as, overlaps with, or is close to the epitope bound by the anti-PD-1 antibody of the present invention. The assay can use ELISA, RIA, BIACORE^TMBiofilm interference or flow cytometry. To test whether an anti-PD-1 antibody cross-competes with another anti-PD-1 antibody, the competition method described above can be used in two directions, i.e., to determine what is knownWhether the antibody blocks the test antibody and vice versa. Such cross-competition experiments may be performed using, for example, the IBIS MX96 SPR instrument or Octet^TMAnd (4) carrying out the system.

The term "chimeric antibody" in its broadest sense refers to an antibody containing one or more regions from one antibody and one or more regions from one or more other antibodies, which are typically antibodies of partially human origin and partially non-human origin (i.e., antibodies derived in part from a non-human animal, e.g., a mouse, rat, or other rodent, or bird such as a chicken). To reduce the risk of human anti-antibody responses (e.g., in the case of murine antibodies, human anti-mouse antibody responses), chimeric antibodies are preferred over non-human antibodies. An example of a typical chimeric antibody is one in which the variable domain sequence is murine and the constant region sequence is human. In the case of chimeric antibodies, the non-human portion may be subjected to further modifications to humanize the antibody. The chimeric antibodies described herein have a chicken variable domain sequence and a human constant region sequence.

The term "humanization" refers to the fact that: in the case where the antibody is of fully or partially non-human origin (e.g., a murine or chicken antibody obtained by immunizing a mouse or chicken, respectively, with an antigen of interest, or a chimeric antibody based on such murine or chicken antibody), it is possible to replace amino acids (particularly in the framework and constant regions of the heavy and light chains) to avoid or minimize immune reactions in humans. While it is not possible to accurately predict immunogenicity and thereby accurately predict human anti-antibody responses to a particular antibody, non-human antibodies tend to be more immunogenic in humans than human antibodies. Chimeric antibodies in which a foreign (e.g., rodent or avian) constant region has been replaced with a human-derived sequence have been shown to be generally less immunogenic than antibodies of entirely foreign origin, and the trend in therapeutic antibodies is toward humanized or fully human antibodies. Chimeric antibodies or other antibodies of non-human origin may thus be humanized to reduce the risk of human anti-antibody reactions.

For chimeric antibodies, humanization typically involves modifying the framework regions of the variable domain sequences. With respect to humanization, amino acid residues that are part of the Complementarity Determining Regions (CDRs) are most often not altered, although in some cases it may be desirable to alter individual CDR amino acid residues, for example to remove glycosylation sites, deamidation sites, aspartate isomerization sites, or unwanted cysteine or methionine residues. N-linked glycosylation occurs by attachment of the oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr (where X can be any amino acid other than Pro). Removal of the N-glycosylation site can be achieved by mutating the Asn or Ser/Thr residues to different residues, preferably by way of conservative substitutions. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, mainly when they are present in the sequence Asn-Gly and to a lesser extent in other dipeptide sequences such as Asn-Ala. When such deamidation sites, in particular Asn-Gly, are present in a CDR sequence, it may therefore be desirable to remove that site, typically by conservative substitution, to remove one of the residues involved.

Many methods for humanizing antibody sequences are known in the art; see, e.g., Almagro&Fransson, Front biosci.13: 1619 summary of 1633 (2008). One commonly used method is CDR grafting, which for example for chimeric antibodies derived from murine involves identifying the human germline gene counterpart to the murine variable domain gene and grafting the murine CDR sequences into this framework. The specificity of the interaction between an antibody and an antigen of interest is mainly found in the amino acid residues located in the six CDRs of the heavy and light chains. Thus, the amino acid sequence within a CDR is more variable between individual antibodies than the sequence outside the CDR. Since the CDR sequences are responsible for the majority of antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody or more generally any particular antibody having a given amino acid sequence, by, for example, constructing an expression vector that expresses CDR sequences from the particular antibody grafted into framework sequences from a different antibody. Thus, it is possible to use non-human antibodies"humanized" and still substantially maintains the binding specificity and affinity of the original antibody. CDR grafting methods can be based on the Kabat CDR definition, although more recent literature (Magdelaine-Beuzelin et al, Crit Rev. Oncol Hematol.64: 210-

Definition (interactive ImmunoGeneTiCs information)

www.imgt.org) can improve the results of humanization (see Lefranc et al, dev. comp immunol.27: 55-77(2003)).

In some cases, CDR grafting may reduce the binding specificity and affinity, and thus the biological activity, of a CDR grafted non-human antibody compared to the parent antibody from which the CDR was obtained. Back mutations (sometimes referred to as "framework repair") can be introduced at selected sites (typically within the framework regions) of the CDR grafted antibody to reestablish the binding specificity and affinity of the parent antibody. Identification of the location of possible back mutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for back-mutation are typically those that are located on the surface of the antibody molecule, while residues that are embedded or have a low degree of surface exposure are not normally altered.

An alternative humanization technique for CDR grafting and back-mutation is resurfacing (resurfacing), in which non-surface exposed residues of non-human origin are retained, while surface residues are changed to human residues.

In certain instances, it may also be desirable to alter one or more CDR amino acid residues to improve binding affinity to a target epitope. This is referred to as "affinity maturation" and optionally may be done in connection with humanization, for example where humanization of an antibody results in a reduction in binding specificity or affinity and the binding specificity or affinity cannot be sufficiently improved by back-mutation alone. Various methods of affinity maturation are known in the art, for example by Burks et al, Proc Natl Acad Sci USA, 94: 412-: 6037-6042 (1998).

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human PD-1, or a portion thereof). It has been shown that certain fragments of full-length antibodies can perform the antigen-binding function of the antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" include (i) Fab fragments: from V_L、V_H、C_LAnd C _H1 domain (e.g., the 12819.17149 and 12865.17150Fab fragments described below); (ii) f (ab')₂Fragment (b): a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) from V_HAnd C _H1 domain; (iv) v from one arm of an antibody_LAnd V_H(iv) an Fv fragment consisting of (V) a dAb fragment consisting of V_HDomain composition; and (vi) an isolated Complementarity Determining Region (CDR) capable of specifically binding to an antigen. Furthermore, although two domains of the Fv fragment (V)_LAnd V_H) Are encoded by separate genes, but they can be recombined by making them able to transform V into V_LAnd V_HThe domain pairs are linked together by a synthetic linker prepared in the form of a single protein chain (known as single chain fv (scfv)) that forms a monovalent molecule. Also falling within the invention are_HAnd/or V_LThe antigen binding molecule of (1). At V_HIn this case, the molecule may also comprise one or more of the CH1, hinge, CH2, or CH3 regions. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies, are also contemplated. The diabody is a bivalent, bispecific antibody, wherein V_HAnd V_LDomains are expressed on a single polypeptide chain, but use a linker that is too short for two domains on the same chain to pair, thereby pairing the domains with complementary domains of another chain and creating two antigen binding sites.

Antibody portions (such as Fab and F (ab')₂Fragments) can be prepared from intact antibodies using conventional techniques, such as papain or pepsin digestion of intact antibodies. In addition, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques (e.g., as described herein).

The class (isotype) and subclass of anti-PD-1 antibodies can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies specific for the class and subclass of the particular antibody. Such antibodies are commercially available. Classes and subclasses can be determined by ELISA, Western blot, and other techniques. Alternatively, class and subclass can be determined by sequencing the entire constant region of the heavy and/or light chain of the antibody, or portions thereof, comparing their amino acid sequences to known amino acid sequences of various immunoglobulin classes and subclasses, and determining the class and subclass of the antibody.

When referring to a particular amino acid residue at a given position of an antibody sequence, an indication such as "35S" refers to both the position and the residue, i.e., in this case it is indicated that a serine residue (S) is present at position 35 of the sequence. Similarly, an indication of, for example, "13Q + 35S" refers to two residues in the corresponding position.

Unless otherwise indicated, all antibody amino acid residue numbers referred to in the disclosure herein are in

Under the numbering scheme.

anti-PD-1 antibodies

The present invention provides antibodies and antigen-binding portions thereof against PD-1. The antibodies may be chimeric, have chicken-derived variable domains and human constant regions, or may be humanized. The antibodies disclosed herein are particularly humanized antibodies.

V of 8 selected humanized anti-PD-1 antibodies of the invention_HAnd V_LThe amino acid sequences (SEQ ID Nos: 2 to 17) are further shown in Table 4 below (example 4). For reference, SEQ ID No. is provided in table 1 below.

The anti-PD-1 antibodies disclosed herein may be referred to with a 5-digit number (e.g., "12819") or with a 10-digit number (e.g., "12819.15384"). As used herein, a 5-digit numbering refers to all antibodies having the heavy and light chain CDR1-3 sequences shown for that digit in table 2, while a 10-digit numbering is used to refer to a particular humanized variant. For example, 12819.15384 is a particular humanized variant having the CDR sequences of 12819 antibody as set forth in table 2. This 5-digit numbering encompasses, for example, antibodies identical to the 10-digit variants shown in table 1 below, except for some alterations in the FR (e.g., lacking residue SY at the N-terminus of the mature light chain, or having residue SS replacing SY). These modifications do not alter the functional (e.g., antigen binding) properties of the antibody.

TABLE 1 amino acid sequences of the heavy and light chain variable domains of humanized anti-PD-1 antibodies SEQ ID Nos

Table 2 below provides the SEQ ID nos of the heavy and light chain CDR amino acid sequences of the antibodies.

TABLE 2 SEQ ID N of CDR amino acid sequences of anti-PD-1 antibodies_o

Antibodies	H-CDR1	H-CDR2	H-CDR3	L-CDR1	L-CDR2	L-CDR3
							12819	18	19	20	21	22	23
12748	24	25	26	27	28	29
							12865	30	31	32	33	34	35
12892	36	37	38	39	40	41
							12796	42	43	44	45	46	47
12777	48	49	50	51	52	53
							12760	54	55	56	57	58	59
13112	60	61	62	63	64	65

In some embodiments, the anti-PD-1 antibody is selected from the group consisting of:

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 18-20;

b) antibodies, variable heavy chain thereofDomain (V)_H) Is identical in sequence to SEQ ID No:2 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 2;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No:2 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 21-23 amino acid sequence;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No:3 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 3;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No:3 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 18-23;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No:2 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No:3 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No:2 and V thereof_LComprises SEQ ID No: 3; and

l) antibody, HC of which comprises SEQ ID No:2 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No:3 and 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 24-26;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 4 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 4;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No: 4 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 27-29;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 5 or 66 is at least 90% identical in amino acid sequence;

g) antibodies, V thereof_LComprises SEQ ID No: 5 or 66;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 5 or 66 and the amino acid sequence of SEQ ID No: 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 24-29;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No: 4 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No: 5 or 66 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 4 and V thereof_LComprises SEQ ID No: 5 or 66; and

l) an antibody whose HC comprises SEQ ID No: 4 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 5 or 66 and SEQ ID No: 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 30-32;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 6 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 6;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No: 6 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 33-35;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 7 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 7;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 7 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 30-35;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No: 6 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No: 7 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 6 and V thereof_LComprises SEQ ID No: 7; and

1) an antibody, HC of which comprises SEQ ID No: 6 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 7 and 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 36-38;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 8 is at least 90% identical in amino acid sequence;

c) antibodies, V thereof_HComprises SEQ ID No: 8;

d) an antibody, the Heavy Chain (HC) of which comprises SEQ ID No: 8 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 39-41;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 9 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 9;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 9 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 36-41;

j) antibodies, V thereof_HIs identical in sequence to SEQ ID No: 8 has an amino acid sequence which is at least 90% identical and which is V_LIs identical in sequence to SEQ ID No: 9 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 8 and V thereof_LComprises SEQ ID No: 9; and

l) antibody, HC of which comprises SEQ ID No: 8 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 9 and 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 42-44;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 10 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 10;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No: 10 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 45-47;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 11 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 11;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 11 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 42-47;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No: 10 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No: 11 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 10 and V thereof_LComprises SEQ ID No: 11; and

1) an antibody, HC of which comprises SEQ ID No: 10 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 11 and 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 48-50 amino acid sequence;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 12 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_MComprises SEQ ID No: 12;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No: 12 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 51-53;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 13 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 13;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 13 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 48-53;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No: 12 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No: 13 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 12 and V thereof_LComprises SEQ ID No: 13; and

l) antibody, HC of which comprises SEQ ID No: 12 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 13 and 68.

a) an antibody having H-CDR1-3 comprising SEQ ID nos: 54-56;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 14 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 14;

d) an antibody whose Heavy Chain (HC) comprises SEQ ID No: 14 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: an amino acid sequence of 57-59;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 15 is at least 90% identical in amino acid sequence;

g) antibodies, V thereof_LComprises SEQ ID No: 15;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 15 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 54-59;

j) antibody, V thereof_HIs identical in sequence to SEQ ID No: 14 has an amino acid sequence which is at least 90% identical and which is V_LIs identical in sequence to SEQ ID No: 15 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 14 and V thereof_LComprises SEQ ID No: 15; and

l) antibody, HC of which comprises SEQ ID No: 14 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 15 and 68.

a) an antibody having a H-CDR1-3 comprising SEQ ID nos: 60-62 amino acid sequence;

b) antibody, its heavy chain variable domain (V)_H) Is identical in sequence to SEQ ID No: 16 is at least 90% identical in amino acid sequence;

c) antibody, V thereof_HComprises SEQ ID No: 16;

d) an antibody, the Heavy Chain (HC) of which comprises SEQ ID No: 16 and 67;

e) an antibody, wherein L-CDR1-3 comprises SEQ ID nos: 63-65;

f) antibody, light chain variable domain (V) thereof_L) Is identical in sequence to SEQ ID No: 17 is at least 90% identical in amino acid sequence;

g) antibody, V thereof_LComprises SEQ ID No: 17;

h) an antibody, the Light Chain (LC) of which comprises SEQ ID No: 17 and 68;

i) an antibody having a H-CDR1-3 and an L-CDR1-3 comprising SEQ ID Nos: 60-65 amino acid sequence;

j) antibodies, V thereof_HIs identical in sequence to SEQ ID No: 16 is at least 90% identical in amino acid sequence and V thereof_LIs identical in sequence to SEQ ID No: 17 is at least 90% identical in amino acid sequence;

k) antibody, V thereof_HComprises SEQ ID No: 16 and V thereof_LComprises SEQ ID No: 17;

1) an antibody, HC of which comprises SEQ ID No: 16 and 67 and LC thereof comprises the amino acid sequence of SEQ ID No: 17 and 68.

In some embodiments, an anti-PD-1 antibody, or antigen-binding portion thereof, comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12819 antibody (e.g., antibody 12819.15384).

In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12748 antibody (e.g., antibody 12748.15381 or antibody 12748.16124).

In some embodiments, an anti-PD-1 antibody, or antigen-binding portion thereof, comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12865 antibody (e.g., antibody 12865.15377).

In some embodiments, an anti-PD-1 antibody, or antigen-binding portion thereof, comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12892 antibody (e.g., antibody 12892.15378).

In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12796 antibody (e.g., antibody 12796.15376).

In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12777 antibody (e.g., antibody 12777.15382).

In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 12760 antibody (e.g., antibody 12760.15375).

In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof comprises the H-CDR1-3 and L-CDR1-3 amino acid sequences of the 13112 antibody (e.g., antibody 13112.15380).

In another embodiment, an anti-PD-1 antibody or antigen-binding portion thereof has a V in amino acid sequence that is identical to any one of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380, respectively_HAnd V_LAt least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the same V_HAnd V_L。

In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof has a V comprising any one of antibodies 12819.15384, 12748.15381, 12748.16124, 12865.15377, 12892.15378, 12796.15376, 12777.15382, 12760.15375, and 13112.15380, respectively_HAnd V_LV of amino acid sequence_HAnd V_L。

In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof comprises the following H-CDR1-3 and L-CDR1-3 amino acid sequences:

a) are respectively SEQ ID No: 18. 19, 20, 21, 22, and 23;

b) are respectively SEQ ID No: 24. 25, 26, 27, 28, and 29;

c) are respectively SEQ ID No: 30. 31, 32, 33, 34, and 35;

d) are respectively SEQ ID No: 36. 37, 38, 39, 40, and 41;

e) are respectively SEQ ID No: 42. 43, 44, 45, 46, and 47;

f) are respectively SEQ ID No: 48. 49, 50, 51, 52, and 53;

g) are respectively SEQ ID No: 54. 55, 56, 57, 58, and 59; or

h) Are respectively SEQ ID No: 60. 61, 62, 63, 64, and 65.

In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises an amino acid sequence that is identical to80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical V_HAnd V that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of V_L：

a) Are respectively SEQ ID No:2 and 3;

b) are respectively SEQ ID No: 4 and 5;

c) are respectively SEQ ID No: 4 and 66;

d) are respectively SEQ ID No: 6 and 7;

e) are respectively SEQ ID No: 8 and 9;

f) are respectively SEQ ID No: 10 and 11;

g) are respectively SEQ ID No: 12 and 13;

h) are respectively SEQ ID No: 14 and 15; or

i) Are respectively SEQ ID No: 16 and 17.

In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof comprises a V comprising an amino acid sequence that is_HAnd V_L：

a) Are respectively SEQ ID No:2 and 3;

b) are respectively SEQ ID No: 4 and 5;

c) are respectively SEQ ID No: 4 and 66;

d) are respectively SEQ ID No: 6 and 7;

e) are respectively SEQ ID No: 8 and 9;

f) are respectively SEQ ID No: 10 and 11;

g) are respectively SEQ ID No: 12 and 13;

h) are respectively SEQ ID No: 14 and 15; or

i) Are respectively SEQ ID No: 16 and 17.

In some embodiments, the anti-PD-1 antibody comprises:

a) consisting of SEQ ID No:2 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 3 and 68;

b) consisting of SEQ ID No: 4 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 5 and 68;

c) consisting of SEQ ID No: 4 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 66 and 68;

d) consisting of SEQ ID No: 6 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 7 and 68;

e) consisting of SEQ ID No: 8 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 9 and 68;

f) consisting of SEQ ID No: 10 and 67 and HC consisting of the amino acid sequences of SEQ ID nos: 11 and 68;

g) consisting of SEQ ID No: 12 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 13 and 68;

h) consisting of SEQ ID No: 14 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 15 and 68; or

i) Consisting of SEQ ID No: 16 and 67 and a HC consisting of the amino acid sequences of SEQ ID nos: 17 and 68.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can have at least one of the following properties:

a) at a K of 750pM or less_DBinds to human PD-1;

b) with a K of 7nM or less_DBinds to cynomolgus monkey PD-1;

c) with a K of 1nM or less_DBinding to mouse PD-1;

d) does not bind to rat PD-1;

e) increased IL-2 secretion in SEB whole blood assay;

f) increasing IFN- γ secretion in a one-way mixed lymphocyte reaction assay;

h) blocks binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 μ g/ml as determined by biofilm interference analysis; and

i) inhibiting tumor growth in vivo.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can have a K of at least 900, at least 850, at least 800, at least 750, at least 700, at least 650, at least 600, at least 550, at least 500, at least 450, at least 400, at least 350, at least 300, at least 250, at least 200, at least 150, at least 100, at least 50, at least 40, at least 30, or at least 20pM_DBinds to human PD-1. In some embodiments, surface plasmon resonance is used to determine K_D. In specific embodiments, the anti-PD-1 antibody or antigen-binding portion is benamazumab, pembrolizumab, or bothHigh affinity binds to human PD-1.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can have a K of at least 9000, at least 8000, at least 7000, at least 6000, at least 5000, at least 4000, at least 3000, at least 2500, at least 2000, at least 1500, at least 1000, at least 900, at least 800, at least 700, at least 600, at least 500, at least 400, at least 300, at least 200, at least 100, at least 75, at least 50, at least 25, at least 20, at least 15, at least 10, or at least 5pM_DBinds to cynomolgus monkey PD-1(SEQ ID No: 89). In some embodiments, surface plasmon resonance is used to determine K_D。

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can have a K of at least 1000, at least 950, at least 900, or at least 850pM_DBinds to mouse PD-1(SEQ ID NO: 91). In some embodiments, surface plasmon resonance is used to determine K_D。

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can inhibit the interaction between PD-1 and PD-L1 by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% in a flow cytometry competition assay at a concentration of 10 μ g/ml. In some embodiments, the anti-PD-1 antibody or antigen-binding portion can inhibit the interaction between PD-1 and PD-L1 by at least 83%.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can block binding of PD-L1 and PD-L2 to PD-1 at a concentration of 10 μ g/ml for at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, as determined by biofilm interferometry. In some embodiments, the anti-PD-1 antibody or antigen-binding portion blocks the binding of PD-L1 and PD-L2 to PD-1 by at least 90%.

In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can compete or cross-compete with 12865, 12892, and 12777 antibodies (e.g., antibodies 12865.15377, 12892.15378, and 12777.15382) for binding to PD-1. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can compete or cross-compete with 12819 antibody (e.g., antibody 12819.15384) for binding to PD-1. In some embodiments, any of the anti-PD-1 antibodies or antigen-binding portions described herein can compete or cross-compete with the 12760 and 13112 antibodies (e.g., antibodies 12760.15375 and 13112.15380) for binding to PD-1.

In some embodiments, an anti-PD-1 antibody (or antigen-binding portion thereof) of the invention binds to a polypeptide comprising SEQ ID No:1 (e.g., at least one, at least two, at least three, at least four, or at least five) of the following residues of 1: v44, V64, L128, P130, K131, A132, E136, and T145. In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1, residues V64, L128, P130, K131, and a132 (such as 12819 antibody, e.g., antibody 12819.15384). In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1, residues K131 and E136 (such as 12865 antibody, e.g., antibody 12865.15377). In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No: residue V44 of 1 and the epitope of PD-1 of T145 bind (such as 13112 antibody, e.g., antibody 13112.15380).

In some embodiments, an anti-PD-1 antibody (or antigen-binding portion thereof) of the invention binds to a polypeptide comprising SEQ ID No:1, residues 56-64, 69-90, and/or 122-140. In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1 and residues 69-90 and 122-140 (such as 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1, residues 56-64, 69-90, and 122-140 (e.g., the 12819 antibody). In some embodiments, the antibody or antigen binding portion binds to a polypeptide comprising SEQ ID No:1 residues 69-90 and 122-140 (e.g., 12865 antibody). In some embodiments, the antibody or portion is identical to SEQ ID No:1 (or a fragment thereof, such as a fragment of one, two, three, four, five, or six residues) to (such as 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In some embodiments, the antibody or portion is identical to SEQ ID No: residue 136-140 of 1 (or a fragment thereof, such as a fragment of one, two, three, or four residues) binds (such as 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). In some embodiments, the antibody or portion is identical to SEQ ID No:1 (or fragments thereof) and residues 136-140 (or fragments thereof) (such as 12819 and 12865 antibodies, e.g., antibodies 12819.15384 and 12865.15377). Epitopes having combinations of any of the above residues are also contemplated.

In some embodiments, an amino acid sequence comprising a PD-1 epitope as described herein can be used as an immunogen (e.g., administered to an animal or as an antigen for screening an antibody library) to generate or identify an anti-PD-1 antibody or antigen-binding portion thereof that binds to the epitope.

The class of anti-PD-1 antibodies obtained by the methods described herein can be altered or converted to another class or subclass. In one aspect of the invention, V is encoded_LOr V_HSuch that it does not comprise a coding C, using methods well known in the art_LOr C_HThe nucleic acid sequence of (1). Code V_LOr V_HThe nucleic acid molecule of (a) is then operably linked to C encoding immunoglobulin molecules from different classes, respectively_LOr C_HThe nucleic acid sequence of (1). This may be done using a catalyst containing C_LOr C_HA vector or nucleic acid molecule for the strand (as described above). For example, anti-PD-1 antibodies, initially IgM, may be class-switched toIgG. Further, class switching may be used to convert one IgG subclass to another, e.g., IgG₁Conversion to IgG₂. The kappa light chain constant region may be changed to a lambda light chain constant region. A preferred method for producing an antibody of the invention having a desired Ig isotype comprises the steps of: isolating a nucleic acid molecule encoding a heavy chain of an anti-PD-1 antibody and a nucleic acid molecule encoding a light chain of an anti-PD-1 antibody, obtaining the variable domain of the heavy chain, linking the variable domain of the heavy chain and the constant region of the heavy chain of a desired isotype, expressing the light chain and the linked heavy chain in a cell, and collecting the anti-PD-1 antibody having the desired isotype.

The anti-PD-1 antibodies of the invention may be IgG, IgM, IgE, IgA, or IgD molecules, but are typically of the IgG isotype, e.g., of the IgG subclass IgG₁、IgG_2aOr IgG_2b、IgG₃Or IgG₄. In one embodiment, the antibody is an IgG₁. In another embodiment, the antibody is an IgG₄。

In one embodiment, the anti-PD-1 antibody may comprise at least one mutation in the Fc region. Several different Fc mutations are known, wherein these mutations provide altered effector function. For example, in many cases, the following may be desirable: reducing or eliminating effector function, for example in the case where ligand/receptor interaction is unwanted or in the case of antibody-drug conjugates.

In one embodiment, the anti-PD-1 antibody comprises at least one mutation in the Fc region that reduces effector function. The amino acid positions of the Fc region that may contribute to the mutation to reduce effector function comprise one or more of positions 228, 233, 234 and 235, wherein the amino acid positions are according to

The numbering plan numbers.

In one embodiment, one or both of the amino acid residues at positions 234 and 235 may be mutated, for example, from Leu to Ala (L234A/L235A). These mutations reduce IgG₁Effector function of the Fc region of an antibody. In addition to and/or atThe amino acid residue at position 228 may, for example, be mutated to Pro. In another embodiment, the amino acid residue at position 233 may be, for example, mutated to Pro, the amino acid residue at position 234 may be, for example, mutated to Val, and/or the amino acid residue at position 235 may be, for example, mutated to Ala. The amino acid position is according to

The numbering plan numbers.

In another embodiment, the antibody is an IgG₄In the case of a subclass, it may comprise the mutation S228P, i.e. having a proline at position 228, wherein the amino acid position is according to

The numbering plan numbers. This mutation is known to reduce unwanted Fab arm exchange.

In some embodiments, an antibody or antigen-binding portion thereof of the invention may be part of a larger immunoadhesion molecule (formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides). Examples of such immunoadhesion molecules include the use of the streptavidin core region to make tetrameric scFv molecules (Kipriyanov et al, Human Antibodies and hybrids 6: 93-101(1995)) and the use of cysteine residues, marker peptides and C-terminal polyhistidine tags to make bivalent and biotinylated scFv molecules (Kipriyanov et al, mol. Immunol.31: 1047-1058 (1994)). Other examples include immunoadhesins (immunoadhesins) in which one or more CDRs from an antibody are incorporated, covalently or non-covalently, into a molecule such that they become specifically bound to an antigen of interest. In such embodiments, the CDRs may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be non-covalently incorporated.

In another embodiment, a fusion antibody or immunoadhesin may be prepared comprising all or part of an anti-PD-1 antibody of the invention linked to another polypeptide. In some embodiments, only the variable domain of the anti-PD-1 antibodyLinked to a polypeptide. In some embodiments, V of an anti-PD-1 antibody_HDomain linked to first polypeptide, and V of anti-PD-1 antibody_LThe domain is linked to a second polypeptide which associates with the first polypeptide in such a way that V_HAnd V_LThe domains can interact with each other to form an antigen binding site. In another preferred embodiment, V_HDomain and V_LThe domains are separated by linkers such that V_HAnd V_LThe domains may interact with each other (e.g., single chain antibodies). V_H-linker-V_LThe antibody is then linked to the polypeptide of interest. Furthermore, a fusion antibody in which two (or more) single-chain antibodies are linked to each other can be created. This is useful if one wants to create a bivalent or multivalent antibody on a single polypeptide chain or if one wants to create a bispecific antibody.

To create Single chain antibodies (scFv), encoding V_HAnd V_LIs operably linked to another fragment encoding a flexible linker, e.g., an amino acid sequence (Gly4-Ser)3, such that V_HAnd V_LThe sequence may be expressed as a continuous single-chain protein, V_LAnd V_HThe domains are connected by flexible linkers. See, e.g., Bird et al, Science 242: 423-426 (1988); huston et al, proc.natl.acad.sci.usa 85: 5879-5883 (1988); and McCafferty et al, Nature 348: 552-554(1990). Single chain antibodies may be monovalent (if only a single V is used)_HAnd V_L) (ii) a Divalent (if two V's are used)_HAnd V_L) (ii) a Or multivalent (if more than two V are used)_HAnd V_L). For example, bispecific or multivalent antibodies can be generated that specifically bind to human PD-1 as well as to another molecule.

In other embodiments, other modified antibodies can be made using nucleic acid molecules encoding anti-PD-1 antibodies. For example, "kappa bodies" (I1l et al, Protein Eng.10: 949-57(1997)), "minibodies ((minibody)" (Martin et al, EMBO J.13: 5303-9(1994)), "diabodies" (Holliger et al, Proc. Natl. Acad. Sci. USA 90: 6444-.

The anti-PD-1 antibodies or antigen-binding portions of the invention can be derivatized or linked to another molecule (e.g., another peptide or protein). In general, the antibody or portion thereof is derivatized such that PD-1 binding is not adversely affected by the derivatization or labeling. Thus, the antibodies and antibody portions of the invention are intended to encompass both intact and modified forms of the human anti-PD-1 antibodies described herein. For example, an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detection agent, a medical agent, and/or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule, such as a streptavidin core region or a polyhistidine tag.

One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to create a bispecific antibody). Suitable crosslinking agents include heterobifunctional crosslinking agents having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional crosslinking agents (e.g., disuccinimidyl suberate). Such linkers are available from, for example, Pierce Chemical Company, Rockford, Il.

anti-PD-1 antibodies can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or carbohydrate groups. These groups may be useful for improving the biological characteristics of the antibody, for example to increase serum half-life.

The antibodies according to the invention may also be labeled. As used herein, the term "label" or "labeling" refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as a polypeptide that incorporates radiolabeled amino acids or is attached to a biotinyl moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). In another embodiment, the label or marker may be therapeutic, such as a drug conjugate or toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β -galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotin-based groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emidine (emetine), mitomycin, etoposide (etoposide), teniposide (tenoposide), vincristine, vinblastine, colchicine, doxorubicin (doruzobicin), and mixtures thereof, Daunorubicin, dihydroxyanthrax-dione, mitoxantrone (mitoxantrone), mithramycin (mithramycin), actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol (propranolol), and puromycin and analogs or homologs thereof. In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

In some embodiments, the antibodies of the invention may be present in a neutral form (including zwitterionic forms) or in a positively or negatively charged species. In some embodiments, the antibody may be complexed with a counterion to form a pharmaceutically acceptable salt.

The term "pharmaceutically acceptable salt" refers to a complex comprising one or more antibodies and one or more counterions, wherein the counterions are derived from pharmaceutically acceptable inorganic and organic acids and bases.

Bispecific binding molecules

In a further aspect, the invention provides bispecific binding molecules having the binding specificity of an anti-PD-1 antibody described herein and the binding specificity of another anti-PD-1 antibody (e.g., another anti-PD-1 antibody described herein) or an antibody that targets a different protein, such as another immune checkpoint protein, a cancer antigen, or another cell surface molecule whose activity mediates a disease condition such as cancer. Such bispecific binding molecules are known in the art, and examples of different types of bispecific binding molecules are given elsewhere herein.

Nucleic acid molecules and vectors

The invention also provides nucleic acid molecules and sequences encoding the anti-PD-1 antibodies or antigen-binding portions thereof described herein. In some embodiments, the different nucleic acid molecules encode the heavy and light chain amino acid sequences of an anti-PD-1 antibody or antigen-binding portion thereof. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain amino acid sequences of an anti-PD-1 antibody, or antigen-binding portion thereof.

Unless otherwise specified, reference to a nucleotide sequence encompasses its complement. Thus, reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand having its complementary sequence. As referred to herein, the term "polynucleotide" means a polymeric form of nucleotides (ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide) that are at least 10 bases in length. The term encompasses single-stranded and double-stranded forms.

The invention also provides nucleotide sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to one or more of the nucleotide sequences recited herein (e.g., to a nucleotide sequence selected from the group consisting of SEQ ID Nos: 69-88). The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues in two sequences that are the same when aligned for maximum correspondence. The length of the sequence identity comparison may be within an extension of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are several different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, a program in Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. FASTA. FASTA (which includes, for example, the programs FASTA2 and FASTA3) provides alignments and percent sequence identities of regions of optimal overlap between query and search sequences (see, for example, Pearson, Methods enzymol.183: 63-98 (1990); Pearson, Methods mol.biol.132: 185-219 (2000); Pearson, Methods enzymol.266: 227-258 (1996); and Pearson, J.mol.biol.276: 71-84 (1998); incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA for its default parameters (word length of 6 and NOPAM factor for scoring matrix) as provided in GCG version 6.1 (incorporated herein by reference) or using Gap for its default parameters as provided in GCG version 6.1.

In one aspect, the invention provides a polypeptide comprising a sequence selected from the group consisting of SEQ ID nos: 69-88.

In any of the above embodiments, the nucleic acid molecule may be isolated.

In a further aspect, the invention provides a vector suitable for expressing one of the chains of an antibody or antigen binding portion thereof as described herein. As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double-stranded segment of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vector (e.g., a non-episomal mammalian vector) can be incorporated into the genome of a host cell upon introduction into the host cell, and thereby is replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The invention provides a vector comprising a nucleic acid molecule encoding a heavy chain of an anti-PD-1 antibody or antigen-binding portion thereof of the invention, a light chain of an anti-PD-1 antibody or antigen-binding portion thereof of the invention, or both a heavy chain and a light chain of an anti-PD-1 antibody or antigen-binding portion thereof of the invention. The invention further provides vectors comprising nucleic acid molecules encoding the fusion proteins, modified antibodies, antibody fragments, and probes thereof.

Nucleic acid molecules encoding the heavy and/or light chain of an anti-PD-1 antibody or antigen-binding portion thereof of the invention can be isolated from any source that produces such antibodies or portions. In various embodiments, the nucleic acid molecule is isolated from a B cell expressing an anti-PD-1 antibody isolated from an animal immunized with a human PD-1 antigen, or from an immortalized cell produced from such a B cell. Methods of isolating nucleic acids encoding antibodies are well known in the art. mRNA can be isolated and used to produce cDNA for use in Polymerase Chain Reaction (PCR) or cDNA cloning of antibody genes. In some embodiments, the nucleic acid molecules of the invention may be synthetic rather than isolated.

In some embodiments, the nucleic acid molecules of the invention may comprise a V encoding an anti-PD-1 antibody or antigen-binding portion from the invention_HA nucleotide sequence of a domain linked in frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecules of the invention may comprise a V encoding an anti-PD-1 antibody or antigen-binding portion from the invention_LA nucleotide sequence of a domain linked in frame to a nucleotide sequence encoding a light chain constant region from any source.

In a further aspect of the invention, the coding heavy chain (V) may be_H) And/or light chain (V)_L) By the variable domain of"full length antibody gene. In one embodiment, code V_HOr V_LThe nucleic acid molecule of the domain is converted to a full-length antibody gene by: inserted into an expression vector which has encoded a heavy chain Constant (CH) or light chain Constant (CL) domain, respectively, such that the V_HSegments are operably connected to CH segments, and/or V, within the carrier_LSegments are operably connected to CL segments within the vector. In another embodiment, code V_HAnd/or V_LThe nucleic acid molecule of the domain is converted to a full-length antibody gene by: encoding V using standard molecular biology techniques_HAnd/or V_LThe nucleic acid molecule of the domain is linked (e.g., linked) to a nucleic acid molecule encoding a CH and/or CL domain. Then, nucleic acid molecules encoding the full-length heavy and/or light chains can be expressed by the cell into which they are introduced and then the anti-PD-1 antibody can be isolated.

The nucleic acid molecules can be used to recombinantly express large amounts of anti-PD-1 antibodies. As described herein, nucleic acid molecules can also be used to produce chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies and antibody derivatives.

In another embodiment, the nucleic acid molecules of the invention are used as probes or PCR primers for specific antibody sequences. For example, the nucleic acid may be used in diagnostic methods as a probe or as a PCR primer to amplify a region of DNA that may be particularly useful for isolating additional nucleic acid molecules encoding the variable domain of an anti-PD-1 antibody. In some embodiments, the nucleic acid molecule is an oligonucleotide. In some embodiments, the oligonucleotides are from the highly variable domains of the heavy and light chains of the antibody of interest. In some embodiments, the oligonucleotide encodes all or part of one or more of the CDRs of an anti-PD-1 antibody or antigen-binding portion thereof of the invention, as described herein.

In another embodiment, the nucleic acid molecules and vectors may be used to prepare mutant anti-PD-1 antibodies. Antibodies may be mutated in the variable domains of the heavy and/or light chains, for example to alter the binding properties of the antibody. For example, mutations can be made in one or more of the CDRs to increase or decrease the K of an anti-PD-1 antibody_DTo therebyIncreasing or decreasing k_offOr to alter the binding specificity of the antibody. In another embodiment, the one or more mutations are made in a monoclonal antibody of the invention at amino acid residues known to be altered compared to the germline. Mutations can be made in the CDR or framework regions of the variable domain or in the constant region. In a preferred embodiment, the mutation is made in the variable domain. In some embodiments, the one or more mutations are made in the CDRs or framework regions of the variable domain of the antibody, or antigen-binding portion thereof, of the invention at amino acid residues known to be altered compared to the germline.

In another embodiment, the framework regions are mutated such that the resulting framework regions have the amino acid sequence of the corresponding germline gene. Mutations may be generated in the framework or constant regions to increase the half-life of the anti-PD-1 antibody. See, for example, PCT publication WO 00/09560. Mutations in the framework or constant regions may also be made to alter the immunogenicity of the antibody and/or to provide sites for covalent or non-covalent binding to another molecule. According to the invention, a single antibody may have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant region.

In some embodiments, an anti-PD-1 antibody or antigen-binding portion thereof of the invention is expressed by: the insertion of DNA encoding partial or full length light and heavy chains (obtained as described above) into an expression vector allows the gene to be operably linked to necessary expression control sequences (such as transcription and translation control sequences). Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. Antibody-encoding sequences may be ligated into vectors such that transcriptional and translational control sequences within the vector exert their intended functions of regulating the transcription and translation of the antibody-encoding sequences. The expression vector and expression control sequences may be selected to be compatible with the expression host cell used. The sequences encoding the antibody light chain and the sequences encoding the antibody heavy chain may be inserted into separate vectors and may be operably linked to the same or different expression control sequences (e.g., promoters). In one embodiment, both coding sequences are inserted into the same expression vector, and may be operably linked to the same expression control sequence (e.g., a common promoter), to separate identical expression control sequences (e.g., promoters), or to different expression control sequences (e.g., promoters). The antibody-encoding sequence can be inserted into the expression vector by standard methods (e.g., ligation of the antibody gene fragment to complementary restriction sites on the vector, or blunt-end ligation in the absence of a restriction site).

Convenient vectors are vectors encoding functionally complete human CH or CL immunoglobulin sequences and having engineered so that any V can be easily inserted and expressed_HOr V_LVector of the appropriate restriction position of the sequence (as described above). The genes encoding HC and LC in such vectors may contain intron sequences that will result in enhanced overall antibody protein production by stabilizing the relevant mRNA. Intron sequences are flanked by splice donor and splice acceptor sites, which determine where RNA splicing can occur. The position of the intron sequence may be in the variable region or the constant region of the antibody chain, or in both the variable region and the constant region when multiple introns are used. Polyadenylation and transcription termination may occur at natural chromosomal sites downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. Antibody chain genes can be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the immunoglobulin chain. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a protein other than an immunoglobulin).

In addition to the antibody chain gene, the recombinant expression vector of the present invention may carry regulatory sequences that control the expression of the antibody chain gene in a host cell. One skilled in the art will appreciate that the design of an expression vector, which comprises the choice of regulatory sequences, may depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, and the like. For mammalian host cell expression, preferred regulatory sequences comprise viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from retroviral LTRs, promoters and/or enhancers derived from Cytomegalovirus (CMV), such as the CMV promoter/enhancer, promoters and/or enhancers derived from simian virus 40(SV40), such as the SV40 promoter/enhancer, promoters and/or enhancers derived from adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma and strong mammalian promoters, such as native immunoglobulin and actin promoters. For further description of viral regulatory elements and their sequences, see, e.g., U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing antibodies in plants (which contain a description of promoters and vectors) and transfection of plants are known in the art. See, for example, U.S. patent 6,517,529. Methods for expressing polypeptides in bacterial cells and fungal cells (e.g., yeast cells) are also well known in the art.

In addition to the antibody chain gene and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically, a selectable marker gene confers resistance to a drug (such as G418, hygromycin or methotrexate) to a host cell into which the vector has been introduced. For example, the selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells in the case of methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthase gene.

As used herein, the term "expression control sequences" means polynucleotide sequences necessary to effect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; high efficiency RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, when desired, sequences that enhance protein secretion. The nature of such control sequences varies with the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequences" is intended to include (at least) all components whose presence is essential for expression and processing, and may also include other components whose presence is beneficial, such as leader sequences and fusion partner sequences.

Host cells and antibodies and methods for producing antibody compositions

Further aspects of the invention are directed to methods for producing the antibody compositions and antibodies and antigen-binding portions thereof of the invention. One embodiment of this aspect of the invention is directed to a method for producing an antibody as defined herein, comprising providing a recombinant host cell capable of expressing the antibody, culturing the host cell under conditions suitable for expression of the antibody, and isolating the resulting antibody. Antibodies produced by such expression in such recombinant host cells are referred to herein as "recombinant antibodies". The invention also provides progeny cells of such host cells and antibodies produced thereby.

As used herein, the term "recombinant host cell" (or just "host cell") means a cell into which a recombinant expression vector has been introduced. The invention provides host cells which may comprise a vector according to the invention, e.g. as described above. The invention also provides a host cell comprising, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portion thereof of an anti-PD-1 antibody or antigen-binding portion thereof of the invention, a nucleotide sequence encoding a light chain or antigen-binding portion thereof of an anti-PD-1 antibody or antigen-binding portion thereof of the invention, or both. It is understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Nucleic acid molecules encoding anti-PD-1 antibodies and vectors comprising these nucleic acid molecules can be used to transfect suitable mammalian, plant, bacterial or yeast host cells. Transfection may be by any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include transfection mediated by dextran, calcium phosphate precipitation, transfection mediated by polybrene (polybrene), protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and microinjection of the DNA directly into the nucleus. In addition, the nucleic acid molecule can be introduced into a mammalian cell by a viral vector. Methods for transforming cells are well known in the art. See, for example, U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art and include, for example, transfection mediated by agrobacterium, biolistic transformation (biolistic transformation), direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines useful as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). These include, inter alia, Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, African green monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell line has a high expression level. Other cell lines that may be used are insect cell lines such as Sf9 or Sf21 cells. When a recombinant expression vector encoding a gene for an antibody is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient for the antibody to be expressed in the host cell or, more preferably, to be secreted into the medium in which the host cell is cultured. The antibody can be recovered from the culture medium using standard protein purification methods. Plant host cells include, for example, tobacco (Nicotiana), Arabidopsis (Arabidopsis), duckweed, maize, wheat, potato, and the like. Bacterial host cells include E.coli and Streptomyces (Streptomyces) species. Yeast host cells include Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Pichia pastoris (Pichia pastoris).

Further, a number of known techniques can be used to enhance expression of the antibodies, or antigen-binding portions thereof, of the invention from a producer cell line. For example, the glutamine synthetase gene expression system (GS system) is a common method for enhancing expression under certain conditions. The GS system is discussed in whole or in part in european patents 0216846, 0256055, 0323997 and 0338841.

It is possible that antibodies expressed from different cell lines or from genetically transgenic animals will have different glycosylation patterns from each other. However, regardless of the glycosylation state of the antibody, and more generally, regardless of the presence or absence of post-translational modifications, all antibodies encoded by or comprising the nucleic acid molecules provided herein are part of the invention.

Pharmaceutical composition

Another aspect of the invention is a pharmaceutical composition comprising as an active ingredient (or as the only active ingredient) an anti-PD-1 antibody or antigen-binding portion thereof or an anti-PD-1 antibody composition of the invention. The pharmaceutical composition may comprise any anti-PD-1 antibody composition or antibody or antigen-binding portion thereof as described herein. In some embodiments, the compositions are intended for use in ameliorating, preventing, and/or treating a disorder associated with PD-1 (e.g., a disorder characterized by overexpression or overactivity of PD-1) and/or cancer. In some embodiments, the composition is intended for use in activating the immune system. In some embodiments, the composition is intended for use in the amelioration, prevention, and/or treatment of cancers originating from tissues such as skin, lung, intestine, ovary, brain, prostate, kidney, soft tissue, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas.

In general, the antibodies of the invention, or antigen binding portions thereof, are suitable for administration as a formulation in combination with one or more pharmaceutically acceptable excipients, for example as described below.

The pharmaceutical compositions of the invention will comprise one or more anti-PD-1 antibodies or binding moieties of the invention, e.g., one or two anti-PD-1 antibodies or binding moieties. In one embodiment, the composition comprises a single anti-PD-1 antibody or binding portion thereof of the invention.

In another embodiment, the pharmaceutical composition may comprise at least one anti-PD-1 antibody or antigen-binding portion thereof (e.g., an anti-PD-1 antibody or portion) and one or more additional antibodies that target one or more associated cell surface receptors (e.g., one or more cancer-associated receptors).

The term "excipient" is used herein to describe any ingredient other than a compound of the invention. The choice of excipients will depend to a large extent on factors such as: the particular mode of administration, the effect of the excipients on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it may be preferable to include isotonic agents (e.g., sugars, polyols such as mannitol, sorbitol, or sodium chloride) in the composition. Other examples of pharmaceutically acceptable substances are wetting or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf-life or effectiveness of the antibody.

The pharmaceutical compositions of the present invention and methods for their preparation will be apparent to those skilled in the art. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19 th edition (Mack Publishing Company, 1995). The pharmaceutical composition is preferably prepared under GMP (good manufacturing practice) conditions.

The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk, in single unit dosage form, or in the form of a plurality of single unit dosages. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject or a convenient fraction of such dose (such as, for example, half or one third of such dose).

Any method recognized in the art for administering peptides, proteins or antibodies may be suitably used for the antibodies and antigen binding portions of the invention.

The pharmaceutical compositions of the present invention are typically suitable for parenteral administration. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by: the tissue of the subject is physically disrupted and the pharmaceutical composition is administered through the breach of the tissue, thus generally resulting in administration directly into the bloodstream, into a muscle, or into an internal organ. Thus, parenteral administration includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through a non-surgical wound penetrating tissue, and the like. In particular, contemplated parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, and intrasynovial injection or infusion; and renal dialysis perfusion techniques. Local perfusion is also contemplated. Preferred embodiments include intravenous and subcutaneous routes.

Formulations of pharmaceutical compositions suitable for parenteral administration typically comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus injection or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oil or aqueous vehicles, pastes, and the like. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. Parenteral formulations also comprise aqueous solutions which may contain excipients such as salts, carbohydrates and buffers (preferably to a pH of 3 to 9), but for some applications they may be more suitably formulated in the form of sterile non-aqueous solutions or dried forms to be used in conjunction with a suitable vehicle (vehicle), such as sterile pyrogen-free water. Exemplary parenteral administration forms include solutions or suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose. Such dosage forms may be suitably buffered, if desired. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form or in liposomal formulation. Formulations for parenteral administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

For example, in one aspect, a sterile injectable solution can be prepared by incorporating the anti-PD-1 antibody or antigen-binding portion thereof or anti-PD-1 antibody composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Proper fluidity of the solution can be maintained, for example, by the use of a coating material, such as lecithin, by the maintenance of the required particle size (in the case of dispersions) and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including agents that delay absorption, such as stearates and gelatins, in the compositions and/or by using modified release coating materials, such as slow release coating materials.

The antibodies of the invention may also be administered intranasally or by inhalation, typically in the form of a dry powder from a dry powder inhaler (alone, in admixture, or as particles of the mixed components, for example, in admixture with a suitable pharmaceutically acceptable excipient), in the form of an aerosol spray from a pressurized container, pump, spray (spray), nebulizer (atomiser), preferably using an electrohydrodynamic nebulizer to produce a fine mist, or atomizer (with or without the use of a suitable propellant), or in the form of nasal drops.

The solution or suspension of the antibody of the invention is typically contained via a pressurized container, pump, spray, nebulizer, or atomizer, which contains a suitable agent, propellant, as a solvent, for example, for dispersing the active, dissolving the active, or extending the release of the active.

Prior to use in dry powder or suspension formulations, the drug product is typically micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any suitable comminution method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

Capsules, tinfoil pouches (blisters) and cartridges (cartridges) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention, a suitable powder base and a property modifying agent.

Suitable solution formulations for nebulizers using electrohydrodynamics to produce fine mist may contain a suitable dose of the antibody of the invention per actuation, and the actuation volume may vary, for example, from 1 to 100 μ L.

Suitable flavouring agents (flavanos), such as menthol and levomenthol, or sweetening agents, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhalation/intranasal administration.

Formulations for inhalation/intranasal administration may be formulated for immediate and/or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

In the case of dry powder inhalers and aerosols, the dosage units are determined by means of a valve delivering a metered amount. Typically, the unit according to the invention is arranged to administer a metered dose or a "puff" of the antibody of the invention. The overall daily dose will typically be administered throughout the day in a single dose or more often in divided doses.

The antibodies and antibody portions of the invention can also be formulated for oral administration. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration, whereby the compound enters the bloodstream directly from the mouth.

Formulations suitable for oral administration include solid, semi-solid and liquid systems, such as tablets; soft or hard capsules containing multi-particles (multi-particles) or nanoparticles (nano-particles), liquids, or powders; lozenges (including liquid-filled); a chew; gelling; a fast-dispersing dosage form; a film; egg-shaped agents (ovules); spraying; and buccal/mucoadhesive patches.

Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be used as fillers in soft or hard capsules (prepared, for example, from gelatin or hydroxypropyl methylcellulose), and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations can also be prepared by reconstitution of solids from, for example, sachets (sachets).

Therapeutic uses of the antibodies and compositions of the invention

In one aspect, the anti-PD-1 antibodies and antigen-binding portions thereof, anti-PD-1 compositions, and bispecific binding molecules of the invention are used to enhance or activate the immune system in a human in need thereof. In some embodiments, the patient has a condition characterized by overexpression or overactivity of PD-1. In some embodiments, the patient is immunosuppressed. In some embodiments, the antibody or antigen-binding portion thereof, composition, or bispecific binding molecule pharmaceutical composition is used to treat cancer, for example, cancer originating from tissues such as skin, lung, intestine, ovary, brain, prostate, kidney, soft tissues, hematopoietic system, head and neck, liver, bladder, breast, stomach, uterus, and pancreas, and any cancer or other condition that is dependent on PD-1 activity or in which the patient expresses or overexpresses PD-L1, PD-L2, or both. Cancers treated by the anti-PD-1 antibodies, antigen-binding portions thereof, anti-PD-1 antibody compositions, and/or bispecific binding molecules of the invention can comprise, for example, melanoma (such as advanced melanoma, or unresectable melanoma or metastatic melanoma), non-small cell lung cancer, bladder cancer, head and neck squamous cell carcinoma, ovarian cancer, colorectal cancer, hodgkin's lymphoma, and Renal Cell Carcinoma (RCC).

In some embodiments, the cancer treated by the anti-PD-1 antibodies, antigen-binding portions, anti-PD-1 compositions, and/or bispecific binding molecules of the invention can comprise, for example, melanoma (e.g., advanced or metastatic melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, hodgkin's lymphoma, non-hodgkin's lymphoma, glioblastoma, glioma, squamous cell lung cancer, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper urinary tract cancer, esophageal cancer, gastroesophageal junction cancer (gastro esophageal junction cancer), gastric cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal cancer, chronic lymphocytic leukemia, acute lymphoblastic leukemia, and/or a combination thereof, Small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, primary peritoneal cancer, fallopian tube cancer, urothelial cancer, T-cell leukemia/lymphoma associated with HTLV, prostate cancer, genitourinary tract cancer, meningioma, adrenocortical cancer, gliosarcoma, fibrosarcoma, renal cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendiceal cancer, biliary tract cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma, and solid tumors.

"treating" refers to a method of reducing or eliminating a biological condition and/or at least one symptom associated therewith. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and/or frequency of symptoms of the disease, disorder, or condition. Further, reference herein to "treatment" includes reference to curative, palliative and prophylactic treatment.

"therapeutically effective amount" refers to an amount of a therapeutic agent administered that will alleviate to some extent one or more of the symptoms of the condition being treated. A therapeutically effective amount of an anti-cancer therapeutic may result in tumor shrinkage, increased survival, elimination of cancer cells, reduction of disease progression, reversal of metastasis, or other clinical endpoint desired by a health care professional.

The antibody composition or antibody or antigen-binding portion thereof of the invention can be administered alone or in combination with one or more other drugs or antibodies (or in any combination thereof). The pharmaceutical compositions, methods and uses of the invention thus also encompass embodiments in combination (co-administration) with other active agents, as detailed below.

As used herein, the terms "co-administration" and "in combination with xx" are intended to mean and do refer to and include the following, in relation to the antibody compositions and antibodies of the invention and antigen-binding portions thereof and one or more other therapeutic agents:

-such combination of antibody composition/antibody/antigen-binding portion and therapeutic agent of the invention is administered simultaneously to a patient in need of treatment, when such components are formulated together in a single dosage form that releases the components to the patient at substantially the same time,

-such a combination of an antibody composition/antibody/antigen-binding portion and a therapeutic agent of the invention is administered to a patient in need of treatment substantially simultaneously, when such components are formulated separately from each other into separate dosage forms, which are taken up by the patient at substantially the same time, after which the components are released to the patient at substantially the same time,

-such a combination of an antibody composition/antibody/antigen-binding portion and a therapeutic agent of the invention is administered sequentially to a patient in need of treatment, when such components are formulated separately from each other in separate dosage forms which are taken up by the patient at successive times with a significant time interval between each administration, after which the components are released to the patient at substantially different times; and

-such a combination of antibody composition/antibody/antigen-binding portion and therapeutic agent of the invention is administered sequentially to a patient in need of treatment, which when such components are formulated together into a single dosage form, releases the components in a controlled manner followed by their simultaneous, sequential, and/or overlapping release to the patient at the same and/or different times,

wherein each moiety may be administered by the same or different route.

The antibody compositions and antibodies and antigen-binding portions thereof of the invention can be administered in the absence of additional therapeutic treatment, i.e., as a separate therapy. Alternatively, treatment with the antibody compositions and antibodies and antigen-binding portions thereof of the invention may comprise at least one additional therapeutic treatment (combination therapy). In some embodiments, the antibody composition or antibody or antigen-binding portion thereof may be co-administered or formulated with another medication/drug for the treatment of cancer. Other therapeutic treatments may include, for example, chemotherapeutic agents, antineoplastic agents, or anti-angiogenic agents, different anti-cancer antibodies, and/or radiation therapy.

The effect can be further improved by combining the antibody composition, antibody, or antigen-binding portion of the present invention with an agent known to induce terminal differentiation of cancer cells. Such compounds may for example be selected from the group consisting of: retinoic acid, trans-retinoic acid, cis-retinoic acid, phenyl butyrate, nerve growth factor, dimethyl sulfoxide, active form vitamin D3, peroxisome proliferator-activated receptor gamma, 12-O-tetradecanoyl phorbol 13-acetate, hexamethylene-bis-acetamide, transforming growth factor beta, butyric acid, cyclic AMP, and vesnarinone. In some embodiments, the compound is selected from the group consisting of retinoic acid, phenyl butyrate, all-trans retinoic acid, and active forms of vitamin D.

A pharmaceutical product comprising an anti-PD-1 antibody composition or an anti-PD-1 antibody or antigen-binding portion thereof of the invention and at least one other agent (e.g., a chemotherapeutic agent, an anti-neoplastic agent, or an anti-angiogenic agent) can be used as a combination therapy for simultaneous, separate, or sequential administration in cancer therapy. The additional agent may be an agent suitable for treating the particular cancer in question, for example an agent selected from the group consisting of: alkylating agents, for example platinum derivatives such as cisplatin, carboplatin and/or oxaliplatin (oxaliplatin); plant alkaloids, such as paclitaxel (paclitaxel), docetaxel (docetaxel) and/or irinotecan (irinotecan); antitumor antibiotics, such as doxorubicin (adriamycin), daunorubicin, epirubicin (epirubicin), idarubicin (idarubicin) mitoxantrone, actinomycin D, bleomycin, actinomycin, luteolin (1uteomycin), and/or mitomycin; topoisomerase inhibitors such as topotecan (topotecan); and/or antimetabolites such as fluorouracil and/or other fluoropyrimidines.

The anti-PD-1 antibodies or antigen-binding portions thereof or anti-PD-1 antibody compositions of the present invention can also be used in combination with other anti-cancer treatments such as vaccines, cytokines, enzyme inhibitors, and T cell therapies. In the case of a vaccine, it may for example be a protein, peptide or DNA vaccine containing one or more antigens associated with the cancer being treated, or a vaccine comprising dendritic cells plus antigen. Suitable cytokines include, for example, IL-2, IFN- γ, and GM-CSF. An example of an enzyme inhibitor with anti-cancer activity is an indoleamine-2, 3-dioxygenase (IDO) inhibitor, e.g. 1-methyl-D-tryptophan (1-D-MT). Adoptive T cell therapy refers to a variety of immunotherapeutic techniques involving expanding or engineering a patient's own T cells to recognize and attack their tumor.

It is also contemplated that the anti-PD-1 antibodies or antigen-binding portions thereof or anti-PD-1 antibody compositions of the invention can be used in adjuvant therapy in combination with a tyrosine kinase inhibitor. These are synthetic, low molecular weight molecules derived primarily from quinazoline (quinazoline) that interact with the intracellular tyrosine kinase domain of the receptor and inhibit ligand-induced receptor phosphorylation by competing for intracellular Mg-ATP binding sites.

In some embodiments, the antibody composition or antibody or antigen-binding portion thereof may be used in combination with another medication/drug that mediates the activation of the immune system, including, but not limited to, agents that mediate the expression or activity of A2AR, BLTA, B7-H3, B7-H4, CTLA-4, CD27, CD28, CD40, CD55, CD73, CD122, CD137, CD160, CGEN-15049, CHK1, CHK2, CTLA-3, CEACAM (e.g., CEACAM-1 and/or CEACAM-5), GAL9, GITR, HVEM, ICOS, IDO, KIR, LAIR1, LAG-3, OX40, TIGIT, TIM-3, TGFR- β, VISTA, and/or 2B 4. In some embodiments, the agent is an antibody or antigen-binding fragment thereof that binds to one of the above molecules. In some embodiments, an antibody composition or antibody or antigen-binding portion thereof of the invention may be administered in combination with a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody, such as tremelimumab (tremelimumab) or yiprimab (ipilimumab)). In one embodiment, an antibody composition or antibody or antigen-binding portion thereof of the invention can be administered in combination with an Yiprimab.

In certain aspects, the antibodies and antigen binding portions of the invention may be administered in combination with another inhibitor of the PD-1 pathway (which may target PD-1 or one or more of its ligands). Examples of such inhibitors include other anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-PD-L2 antibodies. In some embodiments, the antibody compositions, antibodies, and/or antigen-binding portions of the invention may be administered in combination with pembrolizumab and/or nivolumab.

It will be appreciated that the antibody compositions and antibodies and antigen-binding portions thereof of the invention may be used in methods of treatment as described herein, may be used in treatments as described herein, and/or may be used in the manufacture of medicaments for use in treatments as described herein.

Dosage and route of administration

The antibody compositions of the invention will be administered in an effective amount to treat the condition in question, i.e., in dosages and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as: the particular condition being treated, the age, sex, and weight of the patient, and whether the antibody is administered as an independent treatment or in combination with one or more additional anti-cancer treatments.

The dosage regimen may be adjusted to provide the most desirable desired response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be reduced or increased proportionally as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unit doses for patients/subjects for treatment; each unit containing an approximate quantity of active compound calculated to produce the desired therapeutic effect plus a required pharmaceutical carrier. The specification for the dosage unit forms of the invention is generally governed by and directly depends on the following: (a) the unique characteristics of chemotherapeutic agents and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of compounding such active compounds for therapeutic sensitivity in an individual.

Thus, based on the disclosure provided herein, the skilled artisan will appreciate that the dosage and dosing regimen are adjusted according to methods well known in the therapeutic art. That is, the maximum tolerable dose can be readily established and the effective amount to provide the patient with a detectable therapeutic benefit can be determined, as can the time required to administer each dose to provide the patient with a detectable therapeutic benefit. Thus, while certain dosages and administration regimens are exemplified herein, these examples in no way limit the dosages and administration regimens that may be provided to a patient in practicing the invention.

It should be noted that dosage values may vary with the type and severity of the condition being alleviated, and may comprise a single dose or multiple doses. It will be further understood that for a particular subject, a particular dosage regimen should be adjusted over time according to the needs of the individual and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or implementation of the embodied compositions. Further, the dosage regimen using the compositions of the present invention can be based on a variety of factors including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular antibody utilized. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. For example, the dosage may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical efficacy, such as toxicity effects and/or laboratory values. Thus, the present invention encompasses an intra-patient dose escalation (escalation) as determined by the skilled artisan. Determining appropriate dosages and regimens is well known in the relevant art and should be understood to be a consideration of those skilled in the art once provided with the teachings disclosed herein.

It is contemplated that suitable dosages of the antibody compositions of the invention will be in the range of 0.1-100mg/kg, such as about 0.5-50mg/kg, for example about 1-20 mg/kg. The antibody composition may, for example, be administered at a dosage of at least 0.25mg/kg, for example at least 0.5mg/kg, such as at least 1mg/kg, for example at least 1.5mg/kg, such as at least 2mg/kg, for example at least 3mg/kg, such as at least 4mg/kg, for example at least 5 mg/kg; and for example up to at most 50mg/kg, such as up to at most 30mg/kg, for example up to at most 20mg/kg, such as up to at most 15 mg/kg. Administration will generally be repeated at suitable intervals, for example once a week, once every two weeks, once every three weeks, or once every four weeks, for a length of time deemed appropriate by the attending physician (who may increase or decrease the dosage as necessary).

An effective amount for tumor therapy can be measured by its ability to: stabilize disease progression and/or alleviate symptoms in a patient, and preferably reverse disease progression, for example by reducing tumor size. The ability of an antibody or composition of the invention to inhibit cancer can be assessed by in vitro assays (e.g., as described in the examples) and in suitable animal models with predictability for efficacy in human tumors. The appropriate dosage regimen will be selected to provide the optimum therapeutic response in each particular case, for example, as a single bolus or as a continuous infusion, with possible adjustment of the dosage as indicated by the urgency of each case.

Diagnostic uses and compositions

The antibodies of the invention may also be used in diagnostic methods (e.g., in vitro, ex vivo). For example, the antibodies can be used to detect and/or measure the level of PD-1 in a sample (e.g., a tissue sample, or a bodily fluid sample, such as inflammatory exudate, blood, serum, intestinal fluid, saliva, or urine) from a patient. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assay, radioimmunoassay, and immunohistology. The invention further encompasses kits (e.g., diagnostic kits) comprising the antibodies described herein.

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. In case of conflict, the present specification, including definitions, will control.

Generally, the nomenclature used and the techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known in the art and commonly employed. The enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as is commonly done in the art or as described herein.

Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Throughout the specification and embodiments, the words "have" and "comprise", or variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation is not an admission that any of these documents form part of the common general knowledge in the art.

In order that the invention may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

Examples

Example 1: cloning of anti-PD-1 antibodies from chicken B cells

By Symplex^TMModes of antibody discovery techniques autotaxing antibody B cell (ASC) cloning antibody genes derived from chickens was performed. Briefly, ASCs were isolated from lymphoid organs of chickens that had been immunized with PD-1 antigen (in the form of soluble protein antigen and/or in its cell membrane bound form as it is displayed naturally on eukaryotic cells). Staining the ASCs with fluorescently labeled antibodies allows the ASCs to be distinguished from other cells (e.g., T cells, naive B cells, monocytes, etc.) prior to sorting into PCR containers. Single ASC sorting was performed by flow cytometry. Subsequently, Symplex was performed on each sorted B cell as described below^TMProgram to generate a program containing associations V_HAnd V_LThe PCR product of (1).

Performing V on sorted ASC_HAnd V_LLigation of coding sequences to facilitate cognate pairing of sequences. The method utilizes a two-step PCR program based on one-step multiplex overlap extension RT-PCR followed by nested PCR. Using Symplex^TMTechnical connection association V_HAnd V_LThe principle of the sequence is described in WO 2005/042774; WO 2008/104184; WO 2010/022738, and Meijer et al, J Mol Biol 358 (3): 764-72 (2006). Briefly, amplified cognate V's are ligated in a so-called nested PCR step by overlap extension PCR_HAnd V_LA fragment of (a). In subsequent procedures, the PCR products are pooled before cloning into a plasmid vector. This is done in such a way that the cloned DNA fragment encoding the variable domain of the chicken antibody can be usedThe complete chimeric antibody is expressed from a single plasmid expression construct in transfected mammalian cells. As a result, it is possible to screen cell supernatants for chimeric antibodies that exhibit specific binding to the PD-1 antigen.

Materials and methods

Modifying Symplex as described in the above-listed publications^TMTechnique for expanding V from sorted chicken B cells_LAnd V_H. Cloning of the functional expression construct was done in two steps as described below.

And (1). Amplification of V containing pairings in nested PCR reactions_HAnd V_LAmplified PCR products of the fragments. This allows the addition of flanking restriction enzyme recognition sites for ApaI and AvrII at each end. Due to the association V_HAnd V_LThe sequences were paired in a single PCR product from each sorted ASC and cloning of the PCR products was performed after combining all PCR fragments. Plasmid pML392 was constructed to accept Symplex by digesting the corresponding restriction sites ApaI and AvrII^TMAnd (3) PCR products. The resulting ligation of the pooled PCR products and pML392 is shown in FIG. 1. Here, the insertion of the PCR products is carried out with V_HAnd V_LSequences were placed before the human CH1-CH2-CH3 and lambda constant cDNA regions to obtain full-length heavy and light chain reading frames.

And 2. step 2. In the initial construct, the two reading frames encoding the heavy and light chain sequences were placed head to head and separated by a DNA sequence containing the restriction enzyme recognition sites for AscI and NheI. The complete expression construct was obtained by inserting the corresponding AscI/NheI digested dual CMV promoter DNA fragment comprising the 5 '-UTR and the signal peptide between the two 5' ends of the heavy and light chain genes, as depicted in fig. 2.

Example 2: cloning of anti-PD-1 reference antibody analogs

This example briefly explains how to generate the reference analogs of anti-PD-1 antibodies, nivolumab and pembrolizumab.

The amino acid sequences encoding the heavy and light chain variable domains of antibody analogs of nivolumab and pembrolizumab are derived from

Website imgt.org/mAb-DB/acquisition; see table 3 below. Human codon usage was used to reverse translate protein sequences into DNA sequences. The corresponding DNA sequence is then genetically synthesized and cloned into a vector containing a constant human IgG₄Expression of the full-length antibody results in expression of the heavy or kappa light domain in the vector. To prevent Fab arm exchange, the serine residue at position 228 was replaced with proline (Angal et al, mol. Immunol.30: 105-108 (1993)). CHO cells were transfected with the corresponding expression plasmid using standard protein expression systems. The corresponding antibody supernatant was purified using standard protein a purification column chromatography.

TABLE 3 genetically synthesized antibody analogs

Example 3: screening of antibody libraries for binding to PD-1 expressed on the surface of cells

293fectin in 384 well format^TMTransfection reagents (Invitrogen, Cat. No. 12347-019) were individually transfected in HEK293 cells and expressed anti-PD-1 repertoire of clonal antibodies, and antibody-containing supernatants were collected on day 6 post-transfection.

For cell-based antibody screening, FreeStyle was used in 384-well format^TMCHO-S cells were transfected with MAX reagent (Invitrogen, Cat No. 16447-100) to express full-length human PD-1, and untransfected cells were used as a negative control. To allow for a multiplex screening setup, untransfected cells were labeled with CFSE and mixed with unlabeled PD-1 transfected cells at a ratio of 1 to 1 and a density of 1E6 cells each per ml. In 384 well plates, 40 μ L of this cell mixture was mixed with 10 μ L of antibody-containing supernatant and the antibody bound to the cells was revealed by addition of a goat anti-human IgG (H + L) AF647 secondary antibody (Molecular Probes, catalog No. a21445) in the non-wash setting. Using high throughput flow cytometry (

Screener, intellicy) and used

Software analyzed the data by plotting CFSE versus human IgG binding (AF 647). PD-1 specific primary hits were identified as antibody clones that only bound to human PD-1 transfected cells (CSFE negative) but not to control cells (CFSE positive), and plate numbers and plate coordinates were collected for hit picking and subsequent sequence analysis.

Example 4: humanization of anti-PD-1 antibodies

Humanization of the framework regions of chicken anti-PD-1 antibodies was performed to produce antibody molecules with minimal immunogenicity when administered to humans, while substantially retaining the specificity and affinity of the parent chicken antibody.

Materials and methods

Humanization of chicken-derived antibodies was performed using the "CDR grafting" method, which was performed by Jones et al, Nature 321: 522-525(1986) was originally described. First, the variable heavy chain (V) of the antibody is expressed_H) Domains and variable light chains (V)_L) The domain blasts the human IgG database to find the closest human germline genes. This identified human IGHV 3-23X 01(M99660) and IGLV 3-19X 01(X56178) genes, respectively, as being closest to chicken V_HAnd V_LA gene. Similarly, are each V_HAnd V_LSelected human amino acid sequences for humanization of the J gene region were derived from IGHJ 1x 01(J00256) and IGLJ6 x 01 (M18338). In addition, antibody V was aligned against chicken immunoglobulin germline genes_HAnd V_LGenes to identify somatic mutations in framework regions that may play a role in antibody function and/or structure. Such residues may beTo be included in the final humanized antibody gene as so-called "back-mutated" residues. Finally, it is believed that some amino acid positions known to play an important role in antibody structure, stability and function (the so-called "Vernier residues" (Foote and Winter, J Mol biol.224 (2): 487-99(1992)) result in alternative humanized antibody variants comprising human or chicken residues from the corresponding germline.

According to CDR1 and CDR2

Definitions the CDR sequences herein are determined. For the heavy and light chain CDRs 3, the definitions herein comprise an additional amino acid residue (Cys) upstream of the IMGT-CDR3 and an additional amino acid residue downstream (for V)_HCDR3 is Trp for V_LCDR3 is Phe).

The assembly of chicken CDRs and human framework regions was performed by overlap extension PCR. Subjecting the resulting humanized V_HAnd V_LThe PCR products were cloned into expression vectors (plasmids) carrying human heavy and light chain constant regions. To increase the correct cleavage of the signal peptide upstream of the lambda chain, the second amino acid (Ser) of the lambda gene IGLV3.19 is replaced with another amino acid (Tyr) present in other human germline (e.g. IGLV 3.25). The heavy chain sequence contains two "LALA" mutations (L234A/L235A) which are known to reduce the effector function of the Fc region of IgG1 antibodies (Armour et al, Eur J Immunol.29 (8): 2613-24 (1999); and Armour et al, Mol Immunol.40 (9): 585-93 (2003)). The expression vector also contains the necessary regulatory sequences to allow simultaneous expression of the light and heavy chains that assemble the full-length antibody after transfection in mammalian cells.

Results

The final humanized antibody sequences are shown in table 4 below, and the CDR sequence separations are shown in table 5. CDR sequences in the tables are according to

The numbering plan defines.

TABLE 4V of humanized anti-PD-1 antibodies_HAnd V_LSequence of^＊

CDR regions are italicized, underlined, and in bold.

TABLE 5H-CDR sequences and L-CDR sequences of humanized anti-PD-1 antibodies

All humanized antibodies contained the IgG1 "LALA" variant heavy and light chain constant region amino acid sequences shown below.

Heavy chain constant region (SEO ID No: 67):

light chain constant region(SEO ID No：68)：

Example 5: screening of anti-PD-1 antibody candidates

PD-1 is predominantly expressed on the surface of activated T lymphocytes, where it negatively regulates T cell activity. To select the most functional anti-PD-1 antibody candidates, two different in vitro screening systems were established: staphylococcal enterotoxin b (seb) whole blood assay and one-way mixed lymphocyte reaction assay.

Materials and methods

A full set of 69 unique humanized mabs in the IgG1-LALA scaffold format (i.e., with the "LALA" mutation described in example 4) and cloned and humanized as described above were initially screened for functional activity in the SEB whole blood assay. SEB is a super-antigen (super-antigen) that binds to MHC class II molecules and specific V β regions of the T Cell Receptor (TCR) and drives non-specific stimulation of T cells. This results in polyclonal T cell activation/proliferation and release of cytokines including IL-2 and IFN- γ.

To investigate the relevance of the SEB assay for the screening of anti-PD-1 activity, the expression level of PD-1 was investigated for different donors before and after SEB stimulation. PBMCs from six different donors were tested for PD-1 expression by flow cytometry at day 0 and day 3 after SEB stimulation. Relevant lymphocyte gates were set for further analysis.

Based on screening in the SEB whole blood assay, top 10 lead candidates of anti-PD-1 antibodies were identified using blood from at least three different donors. The anti-PD-1 antibody lead candidates were then further titrated to obtain dose-response curves for each individual antibody compared to positive controls (reference analogs of the anti-PD-1 antibody pembrolizumab (Merck) and nivolumab (Bristol-Myers Squibb)); see example 2.

The functionality of the top 10 selected anti-PD-1 antibodies was verified in an alternative in vitro assay, the one-way Mixed Lymphocyte Reaction (MLR) assay. In this assay, Dendritic Cells (DCs) from one donor were co-cultured with CD4+ T cells from another donor to obtain heterotypic antigen-specific stimulation (evoked in 10-15% of all T cells) leading to T cell activation/proliferation and cytokine secretion.

Due to protein stability issues with one of the candidates (12748.15381), an alternative germline sequence was used for this particular antibody. One of the resulting antibodies, 12748.16124, is mentioned below. This variant has a different V than 12748.15381_LSequence but identical V_HSequence (table 1, above).

Results

The data in figure 4 clearly show that the frequency of PD-1 expressing lymphocytes increased in all the donors tested after SEB stimulation. These observations confirm the relevance of this assay for anti-PD-1 antibody screening.

Titration of the most functional anti-PD-1 antibodies in the SEB assay (shown in fig. 5A-I) identified anti-PD-1 lead candidates with functionality similar to or superior to the positive control antibody analogs pembrolizumab and nivolumab. In this assay, whole blood was stimulated with SEB in the presence of the indicated antibodies for 48h, and IL-2 secretion was measured by ELISA after 48 hours. Each data point represents the average of six replicates, with bars indicating SEM.

FIGS. 5A-H show results obtained using humanized anti-PD-1 antibodies. Since aggregation of one of the antibodies [12748.15381] was higher than 5%, alternative frameworks were tested for this antibody. The data in FIG. 5I show similar functionality for the original humanized antibody [12748.15381] and its germline (framework) variant [12748.16124 ].

The functionality of anti-PD-1 antibodies was verified in a one-way MLR assay. In this assay, dendritic cells from two different donors were co-cultured with CD4⁺T cells (ratio 1: 10) and IFN-. gamma.secretion was measured by MesoScale after 5 days. Each data point represents the average of six replicates, with bars indicating SEM. Data obtained from the one-way MLR assay and the data set forth in FIGS. 6A-H are shown and derived from SEData obtained from the B assay are the same for functionality and ranking of anti-PD-1 antibodies. This agreement in data between the different assays provides further confirmation that the selected antibody is functional.

The selected antibodies were derived from two different major epitope bins (epitope bins), indicating that they bound to two different non-overlapping epitopes. All the anti-PD-1 antibodies shown belong to bin 1, except for the 12760 and 13112 antibodies, which belong to bin 2. anti-PD-1 antibodies from silo 1 were found to show the highest functionality in these in vitro assays.

Example 6: flow cytometric analysis of anti-PD-1 antibodies for PD-L1 ligand blocking activity

This example illustrates how a panel of anti-PD-1 antibodies can be tested for ligand blocking activity by performing a flow cytometry competition assay using cell surface expressed PD-1 and fluorescent dye labeled soluble PD-L1.

Materials and methods

PD-L1 ligand blocking activity was studied in a multiplex cell assay in which human and cynomolgus PD-1 were recombinantly expressed on CHO-S cells and binding of the R-PE (R-phycoerythrin) -labeled human PD-L1-Fc chimeric protein was analyzed by flow cytometry. Use of

R-phycoerythrin conjugation kit (Innova Biosciences, UK) commercial recombinant PD-L1-Fc chimeric protein (R-L1-Fc chimeric protein)&DSystems, USA) was conjugated to R-PE. CHO-S cells transiently transfected to express human PD-1 were mixed with CFSE-stained CHO-S cells transiently expressing cynomolgus monkey PD-1. This cell mixture was then incubated with 50. mu.l of anti-PD-1 antibody at 20. mu.g/ml on ice, followed by addition of 50. mu.l of R-PE-labeled PD-L1-Fc (16.4nM final concentration) at about 3.4. mu.g/ml and further incubation for 20min (final anti-PD-1 antibody concentration: 10. mu.g/ml). Bound antibodies were detected using anti-human IgG light chain antibodies conjugated to APCs (allophycocyanins). The binding of PD-L1 and anti-PD-1 antibody was quantified by flow cytometry to detect R-PE and APC fluorescence, respectively.

Results

The results of the competition experiments are presented in FIGS. 7A-B and summarized in Table 6 below. All anti-PD-1 antibodies were tested at a final antibody concentration of 10. mu.g/ml (see above). The three antibodies tested were capable of inhibiting PD-L1 binding by 83% or more, similar to the anti-PD-1 reference antibody lambrolizumab (merck), which is identical to pembrolizumab and included as a positive control. One antibody (12777.13362P) only partially inhibited binding by 69%. One antibody (13112.13208) did not block PD-1 binding. Binding of PD-L1 to PD-1 expressing cells was set to 0% in the presence of the negative control anti-VEGFR 2 antibody ramucirumab (Genentech).

TABLE 6 inhibition of PD-L1 binding in the Presence of anti-PD-1 antibodies

The humanized variants shown in table 6 have the same amino acid sequence as those in table 1 sharing the first 5 digits in their name, except that the variants in table 1 have amino acid residue "SY" at the N-terminus of the light chain. In some embodiments, the SY dipeptide improves signal peptide processing during expression of the antibody light chain. The variants in tables 1 and 6 are expected to have the same functional properties.

Example 7: measurement of PD-1 antibody affinity for human and cynomolgus monkey PD-1ECD antigens

This example demonstrates that most anti-PD-1 antibodies exhibit high picomolar (pM) affinity and good cross-reactivity to both the extracellular domain (ECD) of human PD-1 and the extracellular domain (ECD) of cynomolgus monkey PD-1.

Materials and methods

Kinetic binding analysis of the purified repertoire of anti-PD-1 antibodies was performed on XPR-36 Surface Plasmon Resonance (SPR) biosensors (Bio-Rad, USA). His-tagged human or cynomolgus monkey PD-1ECD antigen was purchased from Acro Biosystems, UK. On sheetBinding kinetics were measured by immobilizing the anti-PD-1 antibody under conditions of valency and maintaining the monovalent PD-1 antigen in solution, as described previously (Canziani et al, Anal Biochem 325 (2): 301-307 (2004)). The lowest possible anti-PD-1 antibody concentration was used to prevent non-specific binding and mass transport limitations. For measurement of antibody kinetics, the anti-PD-1 antibody was adjusted to a concentration of 1.0 μ g/ml and captured on an anti-human IgG Fc surface generated by immobilizing about 1000RU of monoclonal anti-human Fc antibody (Biacore, Denmark). anti-PD-1 antibodies were tested for binding to human or cynomolgus PD-1ECD at 3-fold concentrations ranging from 25nM to 0.31nM, followed by regeneration of the surface with 3M MgCl2 regeneration buffer (Biacore, Denmark). A high flow rate of 20. mu.l/min, an association time of 3.33min and a dissociation time of between 1.5 and 2.75 hours were used. The recorded binding reactions were fitted to a simple Langmuir (Langmuir) 1: 1 binding model for calculation of the binding rate constant (k) using a double reference_onOr ka), dissociation rate constant (k)_offOr kd) and affinity constant (K)_D)。

Results

Binding kinetics are tabulated in table 7 below, which table 7 illustrates that groups of anti-PD-1 antibodies bind to PD-1 with very high affinity in the pM range. All antibodies recognized human PD-1 with higher affinity than nivolumab and pembrolizumab analogs. Highest affinity antibodies [12819.15384]At K of 20pM_DBinds to human PD-1.

TABLE 7 kinetics of binding of anti-PD-1 antibodies to human or cynomolgus monkey PD-1ECD as measured by Surface Plasmon Resonance (SPR)

Example 8: epitope binning of anti-PD-1 antibodies

This example illustrates how PD-1 antibodies are grouped into several epitope bins based on a competition pattern for pairing. Antibodies belonging to different epitope bins recognize different epitopes on the PD-1 ECD.

Method

Paired antibody competition studies were performed by Surface Plasmon Resonance (SPR) analysis using a Continuous Flow Microspotter (CFM) (washch microfluidics, US) in combination with an IBIS MX96 SPR instrument (IBIS Technologies, The Netherlands). At E2S

Surface plasmon resonance imaging analysis was performed on SPR sensors (Ssens BV, The Netherlands). A total of ten anti-PD-1 antibodies (human, IgG1) were diluted to 10. mu.g/ml in 50mM sodium acetate buffer (pH 4.5). Antibody spotting to E2S using a continuous flow micropipette

And conjugated for 15 min. After spotting, will

Placed in an IBIS MX96 biosensor and inactivated with 1M ethanolamine (pH 8.5) for 10 min. After sensor preparation, antibody competition assays were performed using a typical sandwich assay. Monovalent PD-1ECD antigen (nano Biological, China) was diluted in HBS-EP running buffer and injected at a concentration of 50nM and captured by a conjugated array of anti-PD-1 antibodies. Next, a separate injection of each of 10 PD-1 antibodies diluted to 100nM in HBS-EP running buffer was performed to establish an antibody competition mode. After each competition cycle, the sensor surface was regenerated with 10mM glycine HCl buffer (pH 2.0).

Results

The competition pattern for the ten anti-PD-1 antibodies is presented in fig. 8. 12866 and 12807 were found to have no functional activity in cell-based assays, but were still included because they recognized different epitopes. The tested functional anti-PD-1 antibodies were found to bind to two non-overlapping epitope bins. Functional antibodies belonging to epitope bin 1 all cross-block each other and include nivolumab analogs ("Nivo"), pembrolizumab analogs ("Pembro"), 12819, 12892, 12865, and 12777. These antibodies were found to significantly block the binding of PD-L1 and PD-L2. It was found that 12760 and 13112 bind to separate epitope bin 2 because they cross-block each other, but do not block the binding of any antibody from epitope bin 1. As a result, 12760 and 13112 may bind to different sites on PD-1 that do not overlap with the binding sites for the ligands of PD-L1 and PD-L2.

Cross-blocking

functional antibodies

12819, 12865, 12892, 12777, nivolumab and pembrolizumab belonging to epitope bin 1 can be further subdivided into four sub-bins based on competition with 12866 and 12807 (fig. 8). 12819 (bin 1C) was the only antibody that blocked binding of both 12866 and 12807, whereas nivolumab (bin 1D) blocked only 12866 and pembrolizumab (bin 1F) blocked only 12807. The group of antibodies belonging to silo 1E (12865, 12892, and 12777) are unique in that they do not block the binding of 12866 or 12807.

Finally, 12866 (bin 1A) and 12807 (bin 1B) incorporate unique epitope bins. 12866 is blocked by 12819 and nivolumab but not by other anti-PD-1 antibodies, whereas 12807 is blocked by 12819 and pembrolizumab but not by other anti-PD-1 antibodies.

Example 9: measurement of PD-1 antibody interaction reactivity to mouse and rat PD-1ECD antigens

This example demonstrates that anti-PD-1 antibody 12819.15384 cross-reacts strongly with mouse PD-1 but does not bind rat PD-1.

Materials and methods

His-tagged mice and rat PD-1 ECDs were purchased from Sino biologicals. Kinetic binding assays were performed as described in example 7.

Results

The binding kinetics are tabulated in table 8 below. anti-PD-1 antibody 12819.15384 with mouse PD-1 at a K of 809pM_DBound but did not recognize rat PD-1. The affinity for human PD-1ECD was similar to that measured in example 7. Antibody 12865.17150 did not bind to mouse or rat PD-1. Neither nivolumab nor pembrolizumab reference analogs cross-reacted with mouse or rat PD-1 (data not shown).

TABLE 8 binding kinetics of PD-1 antibody 12819.15384 to human, mouse or rat PD-1ECD as measured by Surface Plasmon Resonance (SPR)

^*N.B: are not bonded.

Example 10: analysis of PD-L1 and PD-L2 ligand blocking Activity of PD-1 mAb

This example illustrates the analysis of the PD-L1 or PD-L2 ligand blocking activity by a competition assay using biofilm interferometry for the components of anti-PD-1 antibodies.

Materials and methods

The study of the PD-L1 or PD-L2 ligand blocking activity was performed by biofilm interference (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). Commercial human PD-1Fc fusion protein (Sino Biological) was captured at a concentration of 5. mu.g/ml on an anti-human Fc sensor chip (Fortebio, USA) and

the negative control antibody blocked the residual anti-Fc site. Next, the antigen-coated surface was saturated with an anti-PD-1 antibody at a concentration of 10. mu.g/ml. The ligand blocking activity of PD-L1 or PD-L2 was assessed by incubation with human PD-L1 or PD-L2 Fc fusion protein (Sino Biological) tested at 5. mu.g/ml after saturation of PD-1 with anti-PD-1 antibody.

Results

The results of the competition assay are presented in table 9 below. All antibodies completely blocked both PD-L1 or PD-L2 ligand binding, except antibodies 12760.13169 (which showed no significant blockade of PD-L1 or PD-L2 (26% and 36%, respectively)) and 13112.13208 (which showed no blockade of PD-L1 and showed a weak blockade of PD-L2 (27% and 53%, respectively)). These results are in good agreement with the epitope binning analysis (example 8) and the epitope mapping analysis (example 11) which showed that all antibodies except 12760 and 13112 bind to overlapping epitopes that are mapped to the PD-L1 and PD-L2 binding sites on PD-1, while the 12760 and 13112 antibodies bind to separate PD-1 sites and do not significantly cross-compete with PD-L1 and PD-L2.

TABLE 9 inhibition of PD-L1 and PD-L2 following saturation with anti-PD-1 antibodies

Example 11: epitope mapping of anti-PD-1 antibodies by PD-1 mutagenesis

Antibody epitopes can generally be characterized as linear epitopes (also known as continuous epitopes) or conformational epitopes (also known as discontinuous epitopes). Linear epitopes are defined based on a single contiguous amino acid sequence, whereas conformational epitopes can consist of many smaller discrete linear sequences or a single contact residue. The collection of contact residues clustered at the intermolecular protein interface between antibody and antigen is also known as a hotspot or core epitope (Moreira et al, Proteins 68 (4): 803-12 (2007)). It is currently accepted that most B cell epitopes are discontinuous in nature (Sivalingam and Shepherd, Mol Immunol.51 (3-4): 304-92012(2012), Kringelum et al, Mol Immunol.53 (1-2): 24-34(2013)), with the average epitope spanning 15-22 amino acid residues, of which 2-5 contribute the majority of the binding energy (Sivalingam and Shepherd, supra).

By ranking the binding affinities to 111 different PD-1 mutants, this example demonstrates how the binding epitopes of the 12819 and 12865 antibodies can be divided into linear epitopes and hot spots that differ from those recognized by either banabumab or pembrolizumab.

Method

The human PD-1 receptor consists of an extracellular domain of 268 amino acids (residues 21-288). The extracellular domain spans amino acids 21-170, followed by a transmembrane domain (residues 171-191) and a cytoplasmic domain (residues 192-288). PD-1 belongs to the immunoglobulin superfamily and consists of a two-layer beta sandwich created by the interaction of 8 antiparallel beta strands arranged into two beta sheets with the GFCC' beta strand on one side and the ABED beta strand on the opposite side. The two beta sheets are stabilized by disulfide bonds between residues C54-C123. Human PD-1: the crystal Structure of the human PD-L1 complex is available (PDB 4ZQK), but the C 'D loop between the C' and D β chains and some of the C-terminal sequence after residue 146 are unstructured and absent (PDB 4ZQK, Zak et al, Structure 23 (12): 2341-2348 (2015)). Recently, human PD-1: crystal structure of pembrolizumab Complex (PDB 5JXE, Na et al, Cell Res.2016[ Epub ] Pre-printing]PMID: 27325296). In this structure, the C' D ring is much more ordered and it is shown that the contact residues important for pembrolizumab binding cluster a core epitope on this ring. Currently, no human PD-1: crystal structure of human PD-L2 complex. The NMR structure of human PD-1 in solution shows a high structural similarity to the crystal structure PDB 4ZQK (PDB 2M2D, Cheng et al, J Biol Chem 288 (17): 11771-85 (2013)). Human PD-1 binds human PD-L1 or PD-L2 ligand at a 1: 1 stoichiometry and binding occurs primarily at overlapping binding sites mediated by the GFCC' beta sheet (Cheng et al, J Biol Chem 288 (17): 11771-11785(2013)) (FIG. 9, panels A and B). Human PD-L1 binds to human PD-1 via contact residues V64, N66, Y68 located in the C β chain and G124, I126, L128, a132, I134 and E136 located in the F and G β chains (Zak et al, Structure 23 (12): 2341-8 (2015)). Human PD-L1 and PD-L2 at 8. mu.M and 2. mu.M K, respectively_DBinds to human PD-1 (Cheng et al, supra).

The protein sequence of human PD-1 was downloaded from Uniprot (accession number Q15116; the amino acid sequence is presented as SEQ ID No: 1). Full-length cynomolgus monkey (Macaca fascicularis) protein sequences were downloaded from Uniprot (accession number BOLAJ3_ MACFA (SEQ ID No: 89)). Full-length protein sequences of chicken (Gallus Gallus), mouse (Mus musculus) and Rattus norvegicus (Rattus norvegicus) PD-1 were downloaded from NCBI (XP-422723. (SEQ ID No: 90), NP-032824.1 (SEQ ID No: 91) and XP-006245633.1 (SEQ ID No: 92), respectively). The sequence identity of the different PD-1 extracellular amino acid sequences compared to human PD-1 is shown in Table 10 below.

TABLE 10 comparison of PD-1ECD sequences between species

	Amino acid differences	% sequence identity
			Cynomolgus monkey PD-1ECD	6	96.0
Brown rat PD-1ECD	50	66.7
			Mouse PD-1ECD	57	62.0
Chicken PD-1ECD	73	51.3

By combining from

Resolution measurement ofHuman PD-1: the crystal structure of human PD-L1 complex (PDB 4ZQK) and the NMR structure of APO human PD-1(PDB 2M2D) structural information established a molecular model for human PD-1. The structure PDB 4ZQK was used as the basis for the model, with the missing C' D loop and C-terminal portion of PD-1 provided from the NMR structure. Next, surface exposed amino acid residues are highlighted and 83 individual alanine substitutions (alanine scans) are designed for the surface exposed residues on the human PD-1ECD and 5 different exposed residue positions between human, mouse and rat PD-1 are back mutated to rat PD-1 residues.

To locate linear antibody epitopes in the context of native human PD-1 structures, 23 chimeric proteins were generated in which 10 amino acids in the human PD-1ECD sequence were sequentially exchanged for chicken sequences in a segment of overlapping 5 amino acids. Sequence exchanges were made in the extracellular domain of human PD-1 spanning amino acids 31-146, since the chicken protein sequence outside this segment does not align well with human PD-1 and is omitted.

Synthesis and cloning of PD-1 cDNA encoding the extracellular domain of human PD-1 into a cDNA containing a CMV promoter and a human IgG₁In a vector of Fc sequence (residues P101-K330), resulting in IgG_lFc C-terminal fusion to the cloned PD-1 ECD. Mutated human PD-1Fc fusion constructs were generated by standard PCR and engineering techniques and used ExpicCHO in 2ml culture^TMThe expression system transiently expresses the protein. Human PD-1Fc fusion constructs were harvested after 9 days and the supernatants were tested for binding affinity to anti-PD-1 Fab by Surface Plasmon Resonance (SPR). Culture supernatants containing PD-1 fusion proteins were immobilized on G-a-hu-IgG Fc using a continuous flow microspotter (CFM, Wash Microfluidics, Salt Lake City, US)

(Ssens BV, The Netherlands) for 15 minutes. After spotting, will

Placed in an IBIS MX96 biosensor and captured using a FixIT kit (Ssens BV, The Netherlands)Proteins are immobilized to the surface. Kinetic analysis was performed by applying the so-called kinetic titration series (Karlsson r.2006) in which monomeric Fab fragments of the antibodies of the invention were injected at increasing concentrations of 1nM to 50nM, without applying a surface regeneration step after each antigen injection. Fab binding was performed for 15 min and antigen dissociation for 30 min. The recorded binding reactions were fitted to a simple Langmuir 1: 1 binding model using Scrubber 2 software for calculation of the binding rate constant (k)_onOr ka), dissociation rate constant (k)_offOr k_d) And affinity constant (K)_D)。

Results

The binding affinities of the anti-PD-1 Fab 12819.17149 and 12865.17150 and the reference analogs nivolumab and pembrolizumab were evaluated. 12819.17149 and 12865.17150 are identical to 12819.15384 and 12865.15377 in the VH and VL amino acid sequences, respectively, but are identified by different 10-digit numbering, since the heavy and light chain sequences of each of the first two variants are co-expressed in the host cell on the same plasmid rather than on separate plasmids. Comprises non-PD-L1 and PD-L2 ligand blocking Fab 13112.15380 and

as a control.

All 111 tested PD-1 mutants expressed well. Only three chimeric constructs did not bind to any of the antibodies tested, suggesting that mutations introduced into these three constructs may result in major conformational perturbations that affect binding of all PD-1 antibodies tested. Change in binding affinity of Fab antibodies binding to mutated PD-1 constructs compared to wild type by K_Dmutant/K_DThe ratio (normalized binding affinity) of the wild type is expressed. A summary of linear epitope scans performed by inserting a 10 amino acid chicken sequence into the human PD-1ECD is shown in table 11 below. At least a 5-fold decrease in affinity was used as a cut-off criterion to detect a decrease in binding affinity to the mutated PD-1 construct. In some examples, binding is not detectable for a particular antibody. These constructs are listed as N.B (not to be bound).

Single contact residues were also located by making 83 alanine substitutions or 5 rat back mutations (table 12 below).

A summary of the linear epitopes or contact residues identified for the antibodies tested is presented in table 13. A schematic of the localized binding epitopes shown in density plots on the structure of human PD-1ECD is shown in figure 9.

Analysis showed that the binding epitopes of 12819 and 12865 anti-PD-1 antibodies were significantly different compared to the reference antibodies nivolumab and pembrolizumab (tables 11-13, fig. 9). The core epitope of pembrolizumab (fig. 9, panel C) is located on the C 'β chain and on the C' -D loop. Contact residues/linear epitopes are also found on the C and F β chains, where contact residues of PD-L1 are also present. The core epitope of nivolumab (fig. 9, panel D) is present on the end of the F β chain as well as on the entire G β chain, covering some of the PD-1 contact residues reported to be utilized by human PD-L1. 12819 and 12865 (FIG. 9, panels E and F) are located on the F and G β chains, covering more area than nivolumab and overlapping in this area with all reported contact residues of human PD-L1. 12865 are also very sensitive to mutations at residues 69-75. 12819 also shares a contact residue with pembrolizumab on the C.beta.chain (V64), which has also been reported as a contact residue of human PD-L1. 12819 and 12865 both share linear epitopes that are located to some of the C and C 'beta strands and the C' D loop. No other contact residues were shared between the antibodies tested, except for residue V64. Non-ligand blocking antibody 13112 was shown to be localized to a region further from the PD-L1 and PD-L2 ligand blocking sites by alanine scanning (fig. 9, panel G).

In summary, this example demonstrates that while 12819, 12865, nivolumab, and pembrolizumab bind overlapping epitopes on human PD-1 that can block binding of PD-L1 and PD-L2 ligands, each antibody has a unique binding epitope as demonstrated from competitive binding analysis (epitope binning, example 8), and is shown at the molecular level by using a set of 111 PD-1 mutants to locate individual linear epitopes and contact residues, as summarized in table 13. 12819 is also the only antibody in the anti-PD-1 group studied that cross-reacts with mouse PD-1ECD (of 809 pM)K_DExample 9), highlighting that the binding epitope of this antibody is unique compared to other tested PD-1 antibodies.

Table 12 Fab antibody binding affinities to alanine-scanned human PD-1ECD residues

List by K_Dmutant/K_DNormalized binding of wild type form expression.

TABLE 13 anti-PD-1 antibody binding epitopes identified by use of mutated PD-1Fc fusion constructs

Example 12: 12819 in vivo efficacy of antibodies in four syngeneic murine tumor models

This example demonstrates the in vivo efficacy of the 12819 antibody in four syngeneic murine tumor models.

Method

2x10⁵SalN (fibrosarcoma), 1X10⁶CT26 (colon cancer), 5x10⁶Individual ASB-XIV (lung cancer), or 8x10⁶Individual MC38 (colon cancer) cells were inoculated subcutaneously into the flanks of 6-8 week old female a/J (Sa1N), BALB/cAnNRj (CT26 and ASB-XIV), or C57BL/6(MC38) mice. Tumors were measured two-dimensionally three times per week by calipers and in mm according to the following equation³Tumor volume was calculated: (Width)²x length x 0.5. At 30-50mm³At the mean tumor size of (a), mice were randomized to two groups of ten animals each and treatment was initiated. Mice were treated three times a week for a total of six treatments by intraperitoneal injection of vehicle buffer or monoclonal antibody 12819.17149, followed by an observation period. Antibody treatment was administered at 10 mg/kg. Two-way ANOVA using Bonferroni's multiple comparison test was used to compare tumor volumes at each time point between treatment groups. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad software, Inc.).

Results

The results show profound tumor suppression effects of antibody 12819.17149 in all of the isogenic tumor models tested (P < 0.001 versus vehicle) (fig. 10). Antibody 12819.17149 induced regression of tumor growth in the Sa1N tumor model and resulted in delayed tumor growth in the CT26, MC38 and ASB-XIV tumor models.

⁺ ⁺Example 13: 12819 antibody was administered in the form of menses using a mixture of CD8/CD4T cells and A375 melanoma cells In vivo efficacy in humanized xenograft tumor models

This example demonstrates the 12819 antibody in which human melanoma cell line A375 and purified human CD8 are present⁺And CD4⁺In vivo efficacy in a T cell mixed semi-humanized xenograft tumor model.

Method

Isolation of 4.5x10 from human PBMC donors⁵An individual CD8⁺And CD4⁺T cells and their reaction with 2.05X10⁶Individual a375 (human melanoma) cancer cells were pooled and then inoculated subcutaneously into the flank of 6-8 week old female NODscid mice. Treatment was initiated on the day of tumor inoculation and by intraperitoneal injection of vehicle buffer,

(pembrolizumab) (10mg/kg), or monoclonal antibody 12819.17149(10mg/kg) mice were treated three times per week for six treatments, followed by an observation period. Tumors were measured two-dimensionally three times per week by calipers and in mm according to the following equation³Tumor volume was calculated: (Width)²x length x 0.5. Two-way ANOVA using Bonferroni's multiple comparison test was used to compare tumor volumes at each time point between treatment groups. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad software, Inc.).

Results

In the semi-humanized tumor model, treatment with antibody 12819.17149 resulted in a significant delay in tumor growth (P < 0.001 vs. vehicle), whereas

Tumor growth showed limited effect compared to vehicle treated groups (figure 11).

TABLE 14 List of SEQ ID Nos

Sequence listing

Italics in DNA sequence to indicate cloning position

SEQ ID No:1 (HUMAN PD-1 polypeptide, Uniprot accession number Q15116(PDCD1_ HUMAN))

SEQ ID No:2 (humanised [12819.15384 ]]V_HAmino acid sequence)

SEQ ID No:3 (humanised [12819.15384 ]]V_LAmino acid sequence)

SEQ ID No: 4 (humanised [12748.15381]]And [12748.16124]V_HAmino acid sequence)

SEQ ID No: 5 (humanised [12748.15381]]V_LAmino acid sequence)

SEQ ID No: 6 (humanised [12865.15377 ]]V_HAmino acid sequence)

SEQ ID No: 7 (humanised [12865.15377 ]]V_LAmino acid sequence)

SEQ ID No: 8 (humanised [12892.15378 ]]V_HAmino acid sequence)

SEQ ID No: 9 (humanised [12892.15378 ]]V_LAmino acid sequence)

SEQ ID No: 10 (humanised [12796.15376 ]]V_HAmino acid sequence)

SEQ ID No: 11 (humanised [12796.15376 ]]V_LAmino acid sequence)

SEQ ID No: 12 (humanised [12777.15382 ]]V_HAmino acid sequence)

SEQ ID No: 13 (humanized [12777.15382 ]]V_LAmino acid sequence)

SEQ ID No: 14 (humanised [12760.15375 ]]V_HAmino acid sequence)

SEQ ID No: 15 (humanised [12760.15375 ]]V_LAmino acid sequence)

SEQ ID No: 16 (humanised [13112.15380 ]]V_HAmino acid sequence)

SEQ ID No: 17 (humanized [13112.15380 ]]V_LAmino acid sequence)

SEQ ID No:18 (12819HCDR1 amino acid sequence)

SEQ ID No: 19(12819HCDR2 amino acid sequence)

SEQ ID No: 20(12819HCDR3 amino acid sequence)

SEQ ID No:21 (12819LCDR1 amino acid sequence)

SEQ ID No: 22(12819LCDR2 amino acid sequence)

SEQ ID No: 23(12819LCDR3 amino acid sequence)

SEQ ID No: 24(12748HCDR1 amino acid sequence)

SEQ ID No: 25(12748HCDR2 amino acid sequence)

SEQ ID No: 26(12748HCDR3 amino acid sequence)

SEQ ID No: 27(12748LCDR1 amino acid sequence)

SEQ ID No: 28(12748LCDR2 amino acid sequence)

SEQ ID No: 29(12748LCDR3 amino acid sequence)

SEQ ID No: 30(12865HCDR1 amino acid sequence)

SEQ ID No: 31(12865HCDR2 amino acid sequence)

SEQ ID No: 32(12865HCDR3 amino acid sequence)

SEQ ID No: 33(12865LCDR1 amino acid sequence)

SEQ ID No: 34(12865LCDR2 amino acid sequence)

SEQ ID No: 35(12865LCDR3 amino acid sequence)

SEQ ID No: 36(12892HCDR1 amino acid sequence)

SEQ ID No: 37(12892HCDR2 amino acid sequence)

SEQ ID No: 38(12892HCDR3 amino acid sequence)

SEQ ID No: 39(12892LCDR1 amino acid sequence)

SEQ ID No: 40(12892LCDR2 amino acid sequence)

SEQ ID No: 41(12892LCDR3 amino acid sequence)

SEQ ID No: 42(12796HCDR1 amino acid sequence)

SEQ ID No: 43(12796HCDR2 amino acid sequence)

SEQ ID No: 44(12796HCDR3 amino acid sequence)

SEQ ID No: 45(12796LCDR1 amino acid sequence)

SEQ ID No: 46(12796LCDR2 amino acid sequence)

SEQ ID No: 47(12796LCDR3 amino acid sequence)

SEQ ID No: 48(12777HCDR1 amino acid sequence)

SEQ ID No: 49(12777HCDR2 amino acid sequence)

SEQ ID No: 50(12777HCDR3 amino acid sequence)

SEQ ID No: 51(12777LCDR1 amino acid sequence)

SEQ ID No: 52(12777LCDR2 amino acid sequence)

SEQ ID No: 53(12777LCDR3 amino acid sequence)

SEQ ID No: 54(12760HCDR1 amino acid sequence)

SEQ ID No: 55(12760HCDR2 amino acid sequence)

SEQ ID No: 56(12760HCDR3 amino acid sequence)

SEQ ID No: 57(12760LCDR1 amino acid sequence)

SEQ ID No: 58(12760LCDR2 amino acid sequence)

SEQ ID No: 59(12760LCDR3 amino acid sequence)

SEQ ID No: 60(13112HCDR1 amino acid sequence)

SEQ ID No: 61(13112HCDR2 amino acid sequence)

SEQ ID No: 62(13112HCDR3 amino acid sequence)

SEQ ID No: 63(13112LCDR1 amino acid sequence)

SEQ ID No: 64(13112LCDR2 amino acid sequence)

SEQ ID No: 65(13112LCDR3 amino acid sequence)

SEQ ID No: 66 (humanized [12748.16124]]V_LAmino acid sequence (alternative germline)

SEQ ID No: 67 (heavy chain constant region amino acid sequence)

SEQ ID No: 68 (light chain lambda constant region amino acid sequence)

SEQ ID No: 69 (humanized [12819.15384 ]]V_HDNA sequence)

SEQ ID No: 70 (humanised [12819.15384 ]]V_LDNA sequence)

SEQ ID No: 71 (humanised [12748.15381]]And [12748.16124]V_HDNA sequence)

SEQ ID No: 72 (humanized [12748.15381]]V_LDNA sequence)

SEQ ID No: 73 (humanised [12865.15377 ]]V_HDNA sequence)

SEQ ID No: 74 (humanized [12865.15377 ]]V_LDNA sequence)

SEQ ID No: 75 (humanized [12892.15378 ]]V_HDNA sequence)

SEQ ID No: 76 (humanized [12892.15378 ]]V_LDNA sequence)

SEQ ID No: 77 (humanised [12796.15376 ]]V_HDNA sequence)

SEQ ID No: 78 (humanised [12796.15376 ]]V_LDNA sequence)

SEQ ID No: 79 (humanized [12777.15382 ]]V_HDNA sequence)

SEQ ID No: 80 (humanized [12777.15382 ]]V_LDNA sequence)

SEQ ID No: 81 (humanized [ 12760.15375)]V_HDNA sequence)

SEQ ID No: 82 (humanised [12760.15375 ]]V_LDNA sequence)

SEQ ID No: 83 (humanised [13112.15380 ]]V_HDNA sequence)

SEQ ID No: 84 (humanized [13112.15380 ]]V_LDNA sequence)

SEQ ID No: 85 (humanised [12748.16124]]V_LDNA sequence (alternate germline)

SEQ ID No: 86 (heavy chain constant region genomic DNA sequence, Inclusion of intron)

SEQ ID No: 87 (heavy chain constant region cDNA sequence)

SEQ ID No: 88 (light chain lambda constant region DNA sequence)

SEQ ID No: 89 (cynomolgus monkey PD-1 polypeptide, NCBI accession number BOLAJ3_ MACFA)

SEQ ID No: 90 (Chicken PD-1 polypeptide, NCBI accession number XP _422723.3)

SEQ ID No: 91 (mouse PD-1 polypeptide, NCBI accession number NP-032824.1)

SEQ ID No: 92 (Brown rat PD-1 polypeptide, NCBI accession number XP _006245633.1)

Sequence listing

<110> Xifu Gen Co Ltd

<120> anti-PD-1 antibodies and compositions

<130> 022675.WO052

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<150> 62/236,341

<151> 2015-10-02

<160> 92

<170> PatentIn 3.5 edition

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Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Arg Tyr

20 25 30

Asp Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Gly Asp Ser Asn Lys Met Thr Arg Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Ser Cys Ile Ala Cys Trp Asp Glu Ala Gly Arg Ile Asp

100 105 110

Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Tyr Asp Gly Ser Ser

20 25 30

Tyr Tyr Gly Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val

35 40 45

Ile Tyr Asn Asn Asn Asn Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Tyr Asp Arg Pro Glu

85 90 95

Thr Asn Ser Asp Tyr Val Gly Met Phe Gly Ser Gly Thr Lys Val Thr

100 105 110

Val Leu

<210> 4

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Gly Ile Gly Asn Asp Gly Ser Tyr Thr Asn Tyr Gly Ala Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Asp Ile Arg Ser Arg Asn Asp Cys Ser Tyr Phe Leu Gly Gly

100 105 110

Cys Ser Ser Gly Phe Ile Asp Val Trp Gly Gln Gly Thr Leu Val Thr

115 120 125

Val Ser Ser

130

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Ser Ser Tyr Ser Tyr Gly Trp

20 25 30

Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Glu Ser

35 40 45

Asn Asn Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

50 55 60

Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Gly Asn Ala Asp Ser Ser Ser Gly Ile Phe Gly

85 90 95

Ser Gly Thr Lys Val Thr Val Leu

100

<210> 6

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Asp His

20 25 30

Gly Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Gly Val Ile Asp Thr Thr Gly Arg Tyr Thr Tyr Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Thr Thr Cys Val Gly Gly Tyr Leu Cys Asn Thr Val Gly Ser

100 105 110

Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 7

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Ser Ser Tyr Tyr Gly

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp

35 40 45

Asp Thr Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser

50 55 60

Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp

65 70 75 80

Glu Ala Asp Tyr Tyr Cys Gly Gly Tyr Glu Gly Ser Ser His Ala Gly

85 90 95

Ile Phe Gly Ser Gly Thr Lys Val Thr Val Leu

100 105

<210> 8

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Ser Ser Thr Gly Gly Ser Thr Gly Tyr Gly Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Ser Ile Ser Gly Asp Ala Trp Ser Val Asp Gly Leu Asp Ala

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 9

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Ala Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Tyr Asn Asn

35 40 45

Gln Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly

50 55 60

Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala

65 70 75 80

Asp Tyr Tyr Cys Gly Ser Tyr Asp Ser Ser Ala Val Gly Ile Phe Gly

85 90 95

Ser Gly Thr Lys Val Thr Val Leu

100

<210> 10

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Thr Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Ser Ser Thr Gly Gly Ser Thr Gly Tyr Gly Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Lys Ser Val Ser Gly Asp Ala Trp Ser Val Asp Gly Leu Asp Ala

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Gly Ser Ala Tyr Gly Trp Tyr

20 25 30

Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Tyr Asn Asn

35 40 45

Gln Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly

50 55 60

Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala

65 70 75 80

Asp Tyr Tyr Cys Gly Ser Tyr Asp Ser Ser Ala Val Gly Ile Phe Gly

85 90 95

Ser Gly Thr Lys Val Thr Val Leu

100

<210> 12

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Ser Tyr

20 25 30

Gly Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Val Ile Ser Gly Ser Gly Ile Thr Thr Leu Tyr Ala Pro Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Pro Ser Ile Thr Asp Gly Trp Thr Tyr Gly Gly Ala Trp

100 105 110

Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Asp Gly Ser Tyr Gly Trp Phe

20 25 30

Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Asp Asn Asp

35 40 45

Asn Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly

50 55 60

Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu Ala

65 70 75 80

Asp Tyr Tyr Cys Gly Asn Ala Asp Leu Ser Gly Gly Ile Phe Gly Ser

85 90 95

Gly Thr Lys Val Thr Val Leu

100

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Phe

20 25 30

Asn Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Val

35 40 45

Ala Glu Ile Ser Ser Asp Gly Ser Phe Thr Trp Tyr Ala Thr Ala Val

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Ser Asp Cys Ser Ser Ser Tyr Tyr Gly Tyr Ser Cys Ile Gly

100 105 110

Ile Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 15

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Ile Ser Asp Asp Gly Ser Tyr

20 25 30

Tyr Tyr Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val

35 40 45

Ile Tyr Ile Asn Asp Arg Arg Pro Ser Asn Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Tyr Asp Ser Ser Ala

85 90 95

Gly Val Gly Ile Phe Gly Ser Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 16

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Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asn Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val

35 40 45

Ala Glu Ile Ser Gly Ser Asn Thr Gly Ser Arg Thr Trp Tyr Ala Pro

50 55 60

Ala Val Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Lys Ser Ile Tyr Gly Gly Tyr Cys Ala Gly Gly Tyr Ser

100 105 110

Cys Gly Val Gly Leu Ile Asp Ala Trp Gly Gln Gly Thr Leu Val Thr

115 120 125

Val Ser Ser

130

<210> 17

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Ser Tyr Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Ser Gly Gly Ser Ser Asp Tyr Tyr Gly Trp

20 25 30

Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Tyr Asn

35 40 45

Asn Lys Arg Pro Ser Asp Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser

50 55 60

Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Gly Asn Ala Asp Ser Ser Val Gly Val Phe Gly

85 90 95

Ser Gly Thr Lys Val Thr Val Leu

100

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 18

Gly Phe Thr Phe Thr Arg Tyr Asp

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 19

Ile Gly Asp Ser Asn Lys Met Thr

1 5

<210> 20

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 20

Cys Ala Lys Gly Ser Cys Ile Ala Cys Trp Asp Glu Ala Gly Arg Ile

1 5 10 15

Asp Ala Trp

<210> 21

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 21

Gly Ser Tyr Asp Gly Ser Ser Tyr

1 5

<210> 22

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 22

Asn Asn Asn

1

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 23

Cys Gly Ser Tyr Asp Arg Pro Glu Thr Asn Ser Asp Tyr Val Gly Met

1 5 10 15

Phe

<210> 24

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 24

Gly Phe Thr Phe Ser Asp Tyr Ala

1 5

<210> 25

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 25

Ile Gly Asn Asp Gly Ser Tyr Thr

1 5

<210> 26

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 26

Cys Ala Ser Asp Ile Arg Ser Arg Asn Asp Cys Ser Tyr Phe Leu Gly

1 5 10 15

Gly Cys Ser Ser Gly Phe Ile Asp Val Trp

20 25

<210> 27

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 27

Ser Ser Tyr Ser

1

<210> 28

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 28

Glu Ser Asn

1

<210> 29

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 29

Cys Gly Asn Ala Asp Ser Ser Ser Gly Ile Phe

1 5 10

<210> 30

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 30

Gly Phe Asp Phe Ser Asp His Gly

1 5

<210> 31

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 31

Ile Asp Thr Thr Gly Arg Tyr Thr

1 5

<210> 32

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 32

Cys Ala Lys Thr Thr Cys Val Gly Gly Tyr Leu Cys Asn Thr Val Gly

1 5 10 15

Ser Ile Asp Ala Trp

20

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 33

Gly Ser Ser Ser Tyr

1 5

<210> 34

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 34

Asp Asp Thr

1

<210> 35

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 35

Cys Gly Gly Tyr Glu Gly Ser Ser His Ala Gly Ile Phe

1 5 10

<210> 36

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 36

Gly Phe Asp Phe Ser Ser Tyr Thr

1 5

<210> 37

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 37

Ile Ser Ser Thr Gly Gly Ser Thr

1 5

<210> 38

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 38

Cys Val Lys Ser Ile Ser Gly Asp Ala Trp Ser Val Asp Gly Leu Asp

1 5 10 15

Ala Trp

<210> 39

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 39

Gly Ser Ala

1

<210> 40

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 40

Tyr Asn Asn

1

<210> 41

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 41

Cys Gly Ser Tyr Asp Ser Ser Ala Val Gly Ile Phe

1 5 10

<210> 42

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 42

Gly Phe Asp Phe Ser Ser Tyr Thr

1 5

<210> 43

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 43

Ile Ser Ser Thr Gly Gly Ser Thr

1 5

<210> 44

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 44

Cys Val Lys Ser Val Ser Gly Asp Ala Trp Ser Val Asp Gly Leu Asp

1 5 10 15

Ala Trp

<210> 45

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 45

Gly Ser Ala

1

<210> 46

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 46

Tyr Asn Asn

1

<210> 47

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 47

Cys Gly Ser Tyr Asp Ser Ser Ala Val Gly Ile Phe

1 5 10

<210> 48

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 48

Gly Phe Asp Phe Ser Ser Tyr Gly

1 5

<210> 49

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 49

Ile Ser Gly Ser Gly Ile Thr Thr

1 5

<210> 50

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 50

Cys Thr Arg Ser Pro Ser Ile Thr Asp Gly Trp Thr Tyr Gly Gly Ala

1 5 10 15

Trp Ile Asp Ala Trp

20

<210> 51

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 51

Asp Gly Ser

1

<210> 52

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 52

Asp Asn Asp

1

<210> 53

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 53

Cys Gly Asn Ala Asp Leu Ser Gly Gly Ile Phe

1 5 10

<210> 54

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 54

Gly Phe Thr Phe Ser Thr Phe Asn

1 5

<210> 55

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 55

Ile Ser Ser Asp Gly Ser Phe Thr

1 5

<210> 56

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 56

Cys Ala Lys Ser Asp Cys Ser Ser Ser Tyr Tyr Gly Tyr Ser Cys Ile

1 5 10 15

Gly Ile Ile Asp Ala Trp

20

<210> 57

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 57

Ile Ser Asp Asp Gly Ser Tyr Tyr

1 5

<210> 58

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 58

Ile Asn Asp

1

<210> 59

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 59

Cys Gly Ser Tyr Asp Ser Ser Ala Gly Val Gly Ile Phe

1 5 10

<210> 60

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 60

Gly Phe Thr Phe Ser Ser Tyr Asn

1 5

<210> 61

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 61

Ile Ser Gly Ser Asn Thr Gly Ser Arg Thr

1 5 10

<210> 62

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic peptides "

<400> 62

Cys Ala Lys Ser Ile Tyr Gly Gly Tyr Cys Ala Gly Gly Tyr Ser Cys

1 5 10 15

Gly Val Gly Leu Ile Asp Ala Trp

20

<210> 63

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 63

Ser Ser Asp Tyr

1

<210> 64

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 64

Tyr Asn Asn

1

<210> 65

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic peptides "

<400> 65

Cys Gly Asn Ala Asp Ser Ser Val Gly Val Phe

1 5 10

<210> 66

<211> 104

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polypeptides "

<400> 66

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Ser Gly Gly Ser Ser Tyr Ser Tyr Gly Trp

20 25 30

Phe Gln Gln Lys Pro Gly Gln Ala Pro Val Thr Val Ile Tyr Glu Ser

35 40 45

Asn Asn Arg Pro Ser Asp Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser

50 55 60

Gly Thr Thr Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Gly Asn Ala Asp Ser Ser Ser Gly Ile Phe Gly

85 90 95

Ser Gly Thr Lys Val Thr Val Leu

100

<210> 67

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic polypeptides "

<400> 67

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 68

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polypeptides "

<400> 68

Gly Gln Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser

1 5 10 15

Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp

20 25 30

Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro

35 40 45

Val Lys Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn

50 55 60

Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys

65 70 75 80

Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val

85 90 95

Glu Lys Thr Val Ala Pro Thr Glu Cys Ser

100 105

<210> 69

<211> 379

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 69

ggcgcgccga ggtgcagctg ctggaatctg gaggaggact ggtccagcca ggtggatccc 60

tgcgactgag ctgcgccgct tctggattca cctttacaag atacgacatg gtgtgggtcc 120

gccaggcacc aggaaaggga ctggagtggg tggctggtat cggcgatagt aacaagatga 180

cccgctacgc acctgccgtc aaagggaggg caacaattag tcgggacaac tcaaagaata 240

ctctgtatct gcagatgaat tccctgcgag ctgaggatac agcagtgtac tattgtgcca 300

aaggtagctg catcgcctgt tgggacgaag ctggccgtat tgatgcatgg ggacagggga 360

ctctggtgac cgtctcgag 379

<210> 70

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic polynucleotides "

<400> 70

gctagcctct tacgagctga ctcaggaccc tgcagtgagt gtcgccctgg gccagacagt 60

gagaatcact tgctccggcg gagggagcta cgatggttcc agctactatg gctggtatca 120

gcagaagcca ggacaggcac ctgtgaccgt catctataac aataacaata ggccatctga 180

cattcccgat cggttcagtg gatctagttc agggaacaca gcttctctga ccattacagg 240

agcccaggct gaggacgaag cagattacta ttgtgggtca tacgacaggc cagaaacaaa 300

ttccgattat gtgggaatgt ttggtagcgg cactaaagtc accgtcctag g 351

<210> 71

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 71

ggcgcgccga ggtgcagctg ctggaaagcg gaggaggact ggtccagcca ggtggatctc 60

tgcgactgag ttgcgccgct tcaggcttca cattttctga ctacgccatg aactgggtga 120

ggcaggctcc tggcaaggga ctggagtggg tcgcaggaat cgggaacgat ggaagttaca 180

ctaattatgg agcagccgtg aaggggagag ctactatttc ccgcgacaac agcaaaaata 240

ccctgtacct gcagatgaac tcactgagag ctgaagatac cgcagtgtac tattgtgcct 300

ctgacatcag gagtcggaat gattgctcct atttcctggg agggtgttcc agcggcttta 360

ttgacgtgtg gggtcagggc accctggtca cagtctcgag 400

<210> 72

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 72

gctagcctct tacgagctga cccaggaccc agcagtgtcc gtcgccctgg gccagacagt 60

gagaatcact tgctccggcg gatccagcta cagctatggg tggttccagc agaagcccgg 120

tcaggcccct gtgaccgtca tctatgaaag taacaatagg ccatcagaca ttcccgatcg 180

gttttctggc tctagttcag gaaacacagc tagtctgacc atcacagggg cccaggctga 240

ggacgaagct gattactatt gtggcaatgc agattccagc tctggaattt tcgggtccgg 300

tactaaagtc accgtcctag g 321

<210> 73

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 73

ggcgcgccga ggtgcagctg ctggaatccg gaggaggact ggtccagcca ggtggatccc 60

tgcgactgag ctgcgccgct tctggattcg actttagcga tcacgggatg cagtgggtga 120

gacaggcacc aggcaaggga ctggagtacg tgggtgtcat cgacaccaca ggccgctata 180

catactatgc acctgccgtc aagggcaggg ctaccattag tcgggacaac tcaaaaaata 240

cactgtacct gcagatgaac tctctgaggg ctgaagatac tgcagtgtac tattgcgcca 300

aaactacctg cgtgggaggg tacctgtgca ataccgtcgg aagtatcgat gcttggggac 360

aggggacact ggtgactgtc tcgag 385

<210> 74

<211> 330

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 74

gctagcctcc tacgagctga ctcaggaccc agcagtgagc gtcgccctgg gccagacagt 60

gagaatcact tgctctggcg gagggtccag ctcttactat ggttggtacc agcagaagcc 120

cggccaggct cctgtgaccg tcatctatga cgatacaaac aggccaagtg gaattcccga 180

tcggttctca ggtagttcat ccggcaatac agcttctctg accatcacag gggcccaggc 240

tgaggacgaa gcagattact attgtggtgg ctatgaagga agctctcacg ccgggatttt 300

tggaagtggg actaaagtca ccgtcctagg 330

<210> 75

<211> 376

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 75

ggcgcgccga ggtgcagctg ctggaaagtg gaggaggact ggtccagcca ggtggaagcc 60

tgagactgtc ttgcgccgct agtggcttcg acttttccag ctacaccatg cagtgggtga 120

ggcaggcacc aggcaaggga ctggagtggg tgggcgtcat ctctagtact ggagggtcta 180

ccggatacgg gcctgctgtg aagggaaggg caacaatttc acgggataac tccaaaaata 240

ctctgtatct gcagatgaac agcctgaggg cagaagacac agccgtgtac tattgcgtga 300

aatcaatctc cggagatgcc tggtctgtgg acgggctgga tgcttggggt cagggcaccc 360

tggtcacagt ctcgag 376

<210> 76

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 76

gctagcctca tacgagctga cccaggaccc agcagtgtcc gtcgccctgg gacagacagt 60

gagaatcact tgctccggag gaggatccgc ctacggttgg tatcagcaga agcccggcca 120

ggcacctgtg accgtcatct actataacaa tcagaggcca tctggcattc ccgaccggtt 180

cagtggatcc agctctggga acacagcaag tctgaccatc acaggcgccc aggctgagga 240

cgaagccgat tactattgtg gaagctatga tagttcagct gtggggattt ttggttctgg 300

cactaaagtc accgtcctag g 321

<210> 77

<211> 376

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 77

ggcgcgccga ggtgcagctg ctggaaagtg gaggaggact ggtccagcca ggtggaagcc 60

tgagactgtc ttgcgccgct agtggcttcg acttttccag ctacaccatg cagtgggtga 120

ggcaggcacc aggcaaggga ctggagtggg tgggcgtcat ctctagtact ggagggtcta 180

ccggatacgg gcctgctgtg aagggaaggg caacaatttc acgggataac tccaaaaata 240

ctctgtatct gcagatgaac agcctgaggg cagaagacac agccgtgtac tattgcgtga 300

aatcagtctc cggagatgcc tggtctgtgg acgggctgga tgcttggggt cagggcaccc 360

tggtcacagt ctcgag 376

<210> 78

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 78

gctagcctca tacgagctga cccaggaccc agcagtgtcc gtcgccctgg gccagacagt 60

gagaatcact tgctccggag gaggatccgc ctacggttgg tatcagcaga agcccggcca 120

ggcacctgtg accgtcatct actataacaa tcagaggcca tctgacattc ccgatcggtt 180

cagtggatcc agctctggga acacagcaag tctgaccatc acaggcgccc aggctgagga 240

cgaagccgat tactattgtg gaagctatga tagttcagct gtggggattt ttggttctgg 300

cactaaagtc accgtcctag g 321

<210> 79

<211> 385

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 79

ggcgcgccga ggtgcagctg ctggaatccg gaggaggact ggtccagcca ggtggaagcc 60

tgcgactgtc ttgcgccgct agtggattcg acttttccag ctacggaatg cagtgggtga 120

ggcaggcacc aggcaaggga ctggagtggg tgggcgtcat ctctggaagt gggattacca 180

cactgtacgc acctgccgtc aagggaaggg ctactatctc acgggacaac tctaaaaata 240

cagtgtatct gcagatgaac tccctgagag ctgaagatac cgcagtctac tattgtacac 300

gctcaccctc catcacagac ggctggactt atggaggggc ctggattgat gcttggggtc 360

agggcactct ggtgaccgtc tcgag 385

<210> 80

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 80

gctagccagc tacgagctga cccaggaccc agcagtgtcc gtcgccctgg gccagacagt 60

gagaatcact tgcagtggcg gagatgggtc atacggttgg ttccagcaga agcccggaca 120

ggcccctgtg accgtcatct atgacaacga taataggcca tctgacattc ccgatcggtt 180

tagtggctcc agctctggaa acacagcttc tctgaccatc acaggggccc aggctgagga 240

cgaagctgat tactattgtg gcaatgcaga cctgtccggg ggtattttcg gcagcggaac 300

taaagtcacc gtcctagg 318

<210> 81

<211> 388

<212> DNA

<213> Artificial sequence

<220>

<221> Source

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 81

ggcgcgccga ggtgcagctg ctggaatctg gaggaggact ggtccagcca ggtggatccc 60

tgagactgag ctgcgccgct tctggattca cctttagtac attcaacatg gtgtgggtca 120

ggcaggcacc tggaaaggga ctggagtacg tggctgaaat ctccagcgac ggctctttta 180

catggtatgc aactgccgtc aagggcaggg ccaccattag tcgggataac tcaaaaaata 240

cagtgtacct gcagatgaat tccctgaggg ctgaggacac cgcagtctac tattgcgcaa 300

aatccgattg ttctagttca tactatggat atagctgtat cgggatcatt gacgcttggg 360

gtcagggcac tctggtgacc gtctcgag 388

<210> 82

<211> 339

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 82

gctagcctcc tatgagctga cccaggaccc agcagtgagc gtcgccctgg gccagacagt 60

gagaatcact tgctccggcg gaattagcga cgatggctct tactattacg gatggttcca 120

gcagaagccc ggacaggccc ctgtgaccgt catctatatt aacgacaggc ggccaagtaa 180

tatccccgat aggttttcag ggtccagctc tggtaacaca gcttctctga ccattacagg 240

ggcccaggct gaggacgaag ctgattatta ctgtggctct tacgatagtt cagcaggggt 300

gggtatcttc ggcagtggaa ctaaagtcac cgtcctagg 339

<210> 83

<211> 400

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 83

ggcgcgccga ggtgcagctg ctggaaagtg gaggaggact ggtccagcca ggtggatcac 60

tgagactgtc ctgcgccgcc tccggcttca ccttttccag ctacaacatg ttctgggtgc 120

gccaggcacc aggaaaggga ctggagtttg tcgctgaaat ctctggtagt aatactggaa 180

gccgaacctg gtacgcacct gccgtgaagg gcagggctac aatttctcgg gacaacagta 240

aaaatactct gtatctgcag atgaactctc tgagggctga ggatacagca gtgtactatt 300

gtgcaaaatc aatctacgga gggtattgcg ccggtggcta ttcctgtggt gtgggcctga 360

ttgacgcatg gggacagggg accctggtca cagtctcgag 400

<210> 84

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 84

gctagcctca tacgagctga cccaggaccc agcagtgtcc gtcgccctgg gccagacagt 60

gagaatcact tgcagtggcg gatccagcga ttactatggg tggttccagc agaagcccgg 120

tcaggcccct gtgaccgtca tctactataa caacaagagg ccatctgaca ttcccgatcg 180

gtttagtggc tctagttcag gaaacacagc ctccctgacc attacagggg cccaggctga 240

ggacgaagct gattactatt gtggcaatgc agactccagc gtgggagtct tcgggtctgg 300

tactaaggtg accgtcctag g 321

<210> 85

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic polynucleotides "

<400> 85

gctagcctct tacgagctga ctcagccacc ttccgtgtcc gtgtccccag gacagaccgc 60

aagaatcaca tgcagtggcg gatccagcta ctcatatggg tggttccagc agaagcctgg 120

tcaggccccc gtgacagtca tctatgagag caacaatagg ccttctgaca ttccagaacg 180

gtttagtggc tctagttcag gaaccacagt gactctgacc atcagcgggg tccaggccga 240

ggacgaagct gattactatt gtggcaacgc tgattccagc tctggaattt tcgggtccgg 300

tacaaaagtg actgtcctag g 321

<210> 86

<211> 1606

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 86

ctcgagtgcc tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 60

ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac 120

ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 180

gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 240

ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt 300

tggtgagagg ccagcacagg gagggagggt gtctgctgga agccaggctc agcgctcctg 360

cctggacgca tcccggctat gcagtcccag tccagggcag caaggcaggc cccgtctgcc 420

tcttcacccg gaggcctctg cccgccccac tcatgctcag ggagagggtc ttctggcttt 480

ttccccaggc tctgggcagg cacaggctag gtgcccctaa cccaggccct gcacacaaag 540

gggcaggtgc tgggctcaga cctgccaaga gccatatccg ggaggaccct gcccctgacc 600

taagcccacc ccaaaggcca aactctccac tccctcagct cggacacctt ctctcctccc 660

agattccagt aactcccaat cttctctctg cagagcccaa atcttgtgac aaaactcaca 720

catgcccacc gtgcccaggt aagccagccc aggcctcgcc ctccagctca aggcgggaca 780

ggtgccctag agtagcctgc atccagggac aggccccagc cgggtgctga cacgtccacc 840

tccatctctt cctcagcacc tgaagccgcc gggggaccgt cagtcttcct cttcccccca 900

aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 960

gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1020

aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1080

ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1140

aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaaggtgg gacccgtggg 1200

gtgcgagggc cacatggaca gaggccggct cggcccaccc tctgccctga gagtgaccgc 1260

tgtaccaacc tctgtcccta cagggcagcc ccgagaacca caggtgtaca ccctgccccc 1320

atcccgggag gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta 1380

tcccagcgac atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac 1440

cacgcctccc gtgctggact ccgacggctc cttcttcctc tatagcaagc tcaccgtgga 1500

caagagcagg tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca 1560

caaccactac acgcagaaga gcctctccct gtccccgggt aaatga 1606

<210> 87

<211> 1000

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/note = "description of artificial sequence: synthetic polynucleotides "

<400> 87

ctcgagtgcc tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac 60

ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac 120

ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca 180

gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac 240

ccagacctac atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagagagt 300

tgagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaagccgc 360

cgggggaccg tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg 420

gacccctgag gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt 480

caactggtac gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca 540

gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa 600

tggcaaggag tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac 660

catctccaaa gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg 720

ggaggagatg accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag 780

cgacatcgcc gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc 840

tcccgtgctg gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag 900

caggtggcag caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca 960

ctacacgcag aagagcctct ccctgtcccc gggtaaatga 1000

<210> 88

<211> 325

<212> DNA

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of artificial sequence: synthetic polynucleotides "

<400> 88

cctaggtcag cccaaggcca accccactgt cactctgttc ccgccctcct ctgaggagct 60

ccaagccaac aaggccacac tagtgtgtct gatcagtgac ttctacccgg gagctgtgac 120

agtggcctgg aaggcagatg gcagccccgt caaggcggga gtggagacca ccaaaccctc 180

caaacagagc aacaacaagt acgcggccag cagctacctg agcctgacgc ccgagcagtg 240

gaagtcccac agaagctaca gctgccaggt cacgcatgaa gggagcaccg tggagaagac 300

agtggcccct acagaatgtt cataa 325

<210> 89

<211> 288

<212> PRT

<213> cynomolgus monkey

<400> 89

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Glu Ser Pro Asp Arg Pro Trp

20 25 30

Asn Ala Pro Thr Phe Ser Pro Ala Leu Leu Leu Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Ala Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Arg Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Ala Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Gln Gly Thr Ile Glu Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Ala Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Leu Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Pro Arg Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 90

<211> 304

<212> PRT

<213> Chicken

<400> 90

Met Gly Lys Glu Ala Pro Ser Gly Thr Gly His Arg His Arg Ala Gln

1 5 10 15

Gln Gly Thr Arg Arg Pro Ala Met Ala Leu Gly Thr Ser Arg Thr Met

20 25 30

Trp Asp Ser Thr Glu Ala Ala Leu Val Val Leu Cys Val Leu Leu Leu

35 40 45

Cys Cys Asn Pro Pro Leu Ala Gly Cys His Gln Val Thr Leu Phe Pro

50 55 60

Ala Thr Leu Thr Arg Pro Ala Gly Ser Ser Ala Thr Phe Ile Cys Asn

65 70 75 80

Ile Ser Met Glu Asn Ser Ser Leu Glu Phe Asn Leu Asn Trp Tyr Gln

85 90 95

Lys Thr Asn Asn Ser Asn Pro Gln Lys Ile Ala Gly Ile Ile Arg Asn

100 105 110

Ile Pro Gln Lys Lys Met Glu Lys Tyr Arg Leu Phe Asn Asn Thr Pro

115 120 125

Val Phe Lys Met Glu Ile Leu Asn Leu His Gln Asn Asp Ser Gly Phe

130 135 140

Tyr Tyr Cys Gly Leu Ile Thr Phe Ser Arg Ser Asp Lys Val Val Glu

145 150 155 160

Ser Ser His Ser Gln Leu Val Val Thr Glu Ala Pro Glu Lys Thr Asn

165 170 175

Thr Ile Asp Glu Pro Ser Glu Glu Glu Ser Ser Pro Pro Asp His Ile

180 185 190

Lys Ala Val Leu Leu Gly Thr Leu Leu Leu Ala Gly Val Ile Val Leu

195 200 205

Leu Leu Phe Gly Tyr Ile Ile Ile Asn Asn Arg Arg Ala Asp Val Gln

210 215 220

Lys Pro Ser Ser Gly Asn Thr Leu Ala Glu Val Lys Pro Pro Val Val

225 230 235 240

Pro Val Pro Thr Val Asp Tyr Gly Val Leu Glu Phe Gln Arg Asp Pro

245 250 255

His Ser Gln Val Pro Leu Glu Thr Cys Pro Ala Glu Gln Thr Glu Tyr

260 265 270

Ala Thr Ile Val Phe Pro Glu Glu Lys Pro Ile Thr Pro Glu Arg Gly

275 280 285

Lys Arg His Lys Asp Glu Arg Thr Trp Gln Leu Pro Ser Gln Pro Cys

290 295 300

<210> 91

<211> 288

<212> PRT

<213> mice

<400> 91

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp

20 25 30

Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala

35 40 45

Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met

50 55 60

Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala

65 70 75 80

Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln

85 90 95

Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp

100 105 110

Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val

130 135 140

Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro

145 150 155 160

Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala

165 170 175

Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val

180 185 190

Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp

195 200 205

Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala

210 215 220

Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro

225 230 235 240

Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly

245 250 255

Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln

260 265 270

Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210> 92

<211> 288

<212> PRT

<213> Brown rat

<400> 92

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp

20 25 30

Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala

35 40 45

Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met

50 55 60

Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala

65 70 75 80

Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln

85 90 95

Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp

100 105 110

Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val

130 135 140

Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro

145 150 155 160

Lys Pro Glu Gly Arg Phe Gln Gly Met Val Ile Gly Ile Met Ser Ala

165 170 175

Leu Val Gly Ile Pro Val Leu Leu Leu Leu Ala Trp Ala Leu Ala Val

180 185 190

Phe Cys Ser Thr Ser Met Ser Glu Ala Arg Gly Ala Gly Ser Lys Asp

195 200 205

Asp Thr Leu Lys Glu Glu Pro Ser Ala Ala Pro Val Pro Ser Val Ala

210 215 220

Tyr Glu Glu Leu Asp Phe Gln Gly Arg Glu Lys Thr Pro Glu Leu Pro

225 230 235 240

Thr Ala Cys Val His Thr Glu Tyr Ala Thr Ile Val Phe Thr Glu Gly

245 250 255

Leu Gly Ala Ser Ala Met Gly Arg Arg Gly Ser Ala Asp Gly Leu Gln

260 265 270

Gly Pro Arg Pro Pro Arg His Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

Claims

1. An anti-PD-1 antibody or antigen-binding portion thereof, wherein the antibody comprises the amino acid sequences of H-CDR1-3 and L-CDR1-3 of SEQ ID Nos. 18-20 and 21-23, respectively.

2. An anti-PD-1 antibody or antigen-binding portion thereof, wherein the antibody comprises V of SEQ ID No. 2_HAmino acid sequence and V of SEQ ID No. 3_LAn amino acid sequence.

3. The anti-PD-1 antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody is an IgG.

4. The anti-PD-1 antibody or antigen-binding portion thereof of claim 3, wherein the antibody is an IgG₁。

5. The anti-PD-1 antibody or antigen-binding portion thereof of claim 3, wherein the antibody comprises at least one mutation in the Fc region.

6. The anti-PD-1 antibody or antigen-binding portion thereof of claim 5, wherein

a) The antibody is IgG₁And one or both of the amino acid residues at positions 234 and 235 is mutated to Ala, or

b) The antibody is IgG₄And the amino acid residue at position 228 is mutated to Pro.

7. An anti-PD-1 antibody consisting of a heavy chain amino acid sequence and a light chain amino acid sequence, wherein the heavy chain variable domain and the constant region consist of the amino acid sequences SEQ ID Nos 2 and 67, respectively, and the light chain variable domain and the constant region consist of the amino acid sequences SEQ ID Nos 3 and 68, respectively.

8. The anti-PD-1 antibody or antigen-binding portion thereof of claim 1 or 2, wherein the antibody or antigen-binding portion thereof has at least one of the following properties:

a) at a K of 750pM or less_DBinds to human PD-1;

b) with a K of 7nM or less_DBinds to cynomolgus monkey PD-1;

c) with a K of 1nM or less_DBinding to mouse PD-1;

d) does not bind to rat PD-1;

e) increased IL-2 secretion in SEB whole blood assay;

f) increasing IFN- γ secretion in a one-way mixed lymphocyte reaction assay;

h) blocks binding of PD-L1 and PD-L2 to PD-1 by at least 90% at a concentration of 10 μ g/ml as determined by biofilm interference (Bio-Layer interference) analysis; and

i) inhibiting the growth of fibrosarcoma, colon carcinoma, lung carcinoma and melanoma in vivo.

9. A pharmaceutical composition comprising the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8, and a pharmaceutically acceptable excipient.

10. The pharmaceutical composition of claim 9, further comprising a chemotherapeutic agent.

11. The pharmaceutical composition of claim 9, further comprising an antineoplastic agent.

12. The pharmaceutical composition of claim 9, further comprising an anti-angiogenic agent or a tyrosine kinase inhibitor.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8 and a nucleotide sequence encoding a light chain of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8.

14. A vector comprising the isolated nucleic acid molecule of claim 13, wherein the vector comprises an expression control sequence.

15. A host cell comprising a nucleotide sequence encoding a heavy chain of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8 and a nucleotide sequence encoding a light chain of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8.

16. A method for producing an anti-PD-1 antibody or antigen-binding portion thereof, comprising providing the host cell of claim 13, culturing the host cell under conditions suitable for expression of the anti-PD-1 antibody or antigen-binding portion thereof, and isolating the resulting anti-PD-1 antibody or antigen-binding portion thereof.

17. A bispecific binding molecule having the binding specificity of an anti-PD-1 antibody according to any one of claims 1-8 and the binding specificity of a different antibody, wherein said bispecific binding molecule comprises H-CDR1-3 and L-CDR1-3 with the amino acid sequences SEQ ID NOs 18-20 and 21-23.

18. Use of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8, the pharmaceutical composition of any one of claims 9-12, or the bispecific binding molecule of claim 17, in the manufacture of a medicament for enhancing immunity in a patient.

19. Use of the anti-PD-1 antibody or antigen-binding portion thereof of any one of claims 1-8, the pharmaceutical composition according to any one of claims 9-12, or the bispecific binding molecule according to claim 17, in the manufacture of a medicament for the treatment of fibrosarcoma, colon cancer, lung cancer, or melanoma in a patient.

20. The use of claim 19, wherein the cancer is advanced or metastatic melanoma or non-small cell lung cancer.

21. The use of any one of claims 18-20, wherein the medicament further comprises a chemotherapeutic agent.

22. The use of any one of claims 18-20, wherein the medicament further comprises an anti-neoplastic agent.

23. The use of any one of claims 18-20, wherein the medicament further comprises an anti-angiogenic agent, or a tyrosine kinase inhibitor.